Patent Publication Number: US-2002006638-A1

Title: Methods for recombinant microbial production of fusion proteins and BPI-derived peptides

Description:
BACKGROUND OF THE INVENTION  
       [0001] The present invention relates generally to methods for recombinant microbial production of fusion proteins and peptides derived from or based on Domain I (amino acids 17-45), Domain II (amino acids 65-99) and Domain m (amino acids 142-169) of bactericidal/permeability-increasing protein (BPI).  
       [0002] BPI is a protein isolated from the granules of mammalian polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microorganisms. Human BPI protein has been isolated from PMNs by acid extraction combined with either ion exchange chromatography [Elsbach,  J. Biol. Chem.,  254:11000 (1979)] or  E. coli  affinity chromatography [Weiss, et al.,  Blood,  69:652 (1987)]. BPI obtained in such a manner is referred to herein as natural BPI and has been shown to have potent bactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein have been reported in FIG. 1 of Gray et al.,  J. Biol. Chem.,  264:9505 (1989), incorporated herein by reference. The Gray et al. DNA and amino acid sequences are set out in SEQ ID NOS: 264 and 265 hereto.  
       [0003] BPI is a strongly cationic protein. The N-terminal half of BPI accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of -3. [Elsbach and Weiss (1981), supra.] A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD has an amphipathic character, containing alternating hydrophobic and hydrophilic regions. This N-terminal fragment of human BPI possesses the anti-bacterial efficacy of the naturally-derived 55 kD human BPI holoprotein. [Ooi et al.,  J. Bio. Chem.,  262: 14891-14894 (1987)]. In contrast to the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly detectable anti-bacterial activity against gram-negative organisms. [Ooi et al.,  J. Exp. Med.,  174:649 (1991).] An N-terminal BPI fragment of approximately 23 kD, referred to as “rBPI 23 ,” has been produced by recombinant means and also retains anti-bacterial activity against gram-negative organisms [Gazzano-Santoro et al.,  Infect. Immun.  60:4754-4761 (1992)]. In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI 23 ). The vector was constructed to encode the 31-residue signal sequence and the first 199 amino acids of the N-terminus of the mature human BPI, as set out in SEQ ID NOS: 264 and 265 taken from Gray et al., supra, except that valine at position 151 is specified by GTG rather than GTC and residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG). Recombinant holoprotein, also referred to as rBPI, has also been produced having the sequence set out in SEQ ID NOS: 264 and 265 taken from Gray et al., supra, with the exceptions noted for rBPI 23 . An N-terminal fragment analog designated rBPI 21  or rBPI 21 Δcys has been described in co-owned, copending U.S. Pat. No. 5,420,019 which is incorporated herein by reference. This analog comprises the first 193 amino acids of BPI holoprotein as set out in SEQ ID NOS: 264 and 265 but wherein the cysteine at residue number 132 is substituted with alanine, and with the exceptions noted for rBPI 23 .  
       [0004] The bactericidal effect of BPI has been reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss,  Inflammation: Basic Principles and Clinical Correlates , eds. Gallin et al., Chapter 30, Raven Press, Ltd. (1992). BPI is commonly thought to be non-toxic for other microorganisms, including yeast, and for higher eukaryotic cells. Elsbach and Weiss (1992), supra, reported that BPI exhibits anti-bacterial activity towards a broad range of gram-negative bacteria at concentrations as low as 10 −8  to 10 −9  M, but that 100- to 1,000-fold higher concentrations of BPI were non-toxic to all of the grain-positive bacterial species, yeasts, and higher eukaryotic cells tested at that time. It was also reported that BPI at a concentration of 10 −6  M or 160 μg/mL had no toxic effect, when tested at a pH of either 7.0 or 5.5, on the gram-positive organisms  Staphylococcus aureus  (four strains),  Staphylococcus epidermidis, Streptococcus faecalis, Bacillus subtilis, Micrococcus lysodeikiticus , and  Listeria monocytogenes . BPI at 10 −6  M reportedly had no toxic effect on the fungi  Candida albicans  and  Candida parapsilosis  at pH 7.0 or 5.5, and was non-toxic to higher eukaryotic cells such as human, rabbit and sheep red blood cells and several human tumor cell lines. See also Elsbach and Weiss,  Advances in Inflammaion Research , ed. G. Weissmann, Vol. 2, pages 95-113 Raven Press (1981). This reported target cell specificity was believed to be the result of the strong attraction of BPI for lipopolysaccharide (LPS), which is unique to the outer membrane (or envelope) of gram-negative organisms.  
       [0005] The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through hydrophobic and electrostatic interactions between the cationic BPI protein and negatively charged sites on the bacterial LPS. Bacterial LPS has been referred to as “endotoxin” because of the potent inflammatory response that is stimulated, i.e., the release of mediators by host inflammatory cells which may ultimately result in irreversible endotoxic shock. BPI binds to lipid A, reported to be the most toxic and most biologically active component of bacterial LPS.  
       [0006] In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS structure, leading to activation of bacterial enzymes that degrade phospholipids and peptidoglycans, altering the permeability of the cell&#39;s outer membrane, and initiating events that ultimately lead to cell death. [Elsbach and Weiss (1992), supra]. BPI is thought to act in two stages. The first is a sublethal stage that is characterized by immediate growth arrest, permeabilization of the outer membrane and selective activation of bacterial enzymes that hydrolyze phospholipids and peptidoglycans. Bacteria at this stage can be rescued by growth in serum albumin supplemented media [Mannion et al.,  J. Clin. Invest.,  85:853-860 (1990)]. The second stage, defined by growth inhibition that cannot be reversed by serum albumin, occurs after prolonged exposure of the bacteria to BPI and is characterized by extensive physiologic and structural changes, including apparent damage to the inner cytoplasmic membrane.  
       [0007] Initial binding of BPI to bacterial LPS leads to organizational changes that probably result from binding to the anionic groups in the KDO region of LPS, which normally stabilize the outer membrane through binding of Mg ++  and Ca ++ . Attachment of BPI to the outer membrane of gram-negative bacteria produces rapid permeabilization of the outer membrane to hydrophobic agents such as actinomycin D. Binding of BPI and subsequent gram-negative bacterial killing depends, at least in part, upon the LPS polysaccharide chain length, with long O-chain bearing, “smooth” organisms being more resistant to BPI bactericidal effects than short O-chain bearing, “rough” organisms [Weiss et al.,  J. Clin. Invest.  65: 619-628 (1980)]. This first stage of BPI action, permeabilization of the gram-negative outer envelope, is reversible upon dissociation of the BPI, a process requiring the presence of divalent cations and synthesis of new LPS [Weiss et al.,  J. Immunol.  132: 3109-3115 (1984)]. Loss of gram-negative bacterial viability, however, is not reversed by processes which restore the envelope integrity, suggesting that the bactericidal action is mediated by additional lesions induced in the target organism and which may be situated at the cytoplasmic membrane [Mannion et al.,  J. Clin. Invest.  86: 631-641 (1990)]. Specific investigation of this possibility has shown that on a molar basis BPI is at least as inhibitory of cytoplasmic membrane vesicle function as polymyxin B [In&#39;t Veld et al.,  Infection and Immunity  56: 1203-1208 (1988)] but the exact mechanism as well as the relevance of such vesicles to studies of intact organisms has not yet been elucidated.  
       [0008] In addition to its direct bactericidal activity, BPI is also capable of neutralizing the endotoxic properties of living or dead bacteria and LPS released from the bacteria. Because of its gram-negative bactericidal properties and its ability to bind to and neutralize bacterial LPS, BPI can be utilized for the treatment of mammals suffering from diseases caused by gram-negative bacteria, including bacteremia, endotoxemia, and sepsis. These dual properties of BPI make BPI particularly useful and advantageous for such therapeutic administration.  
       [0009] BPI protein products, including BPI-derived peptides, are useful as adjunct therapy with conventional antibiotics as described in copending and co-assigned U.S. patent application Ser. No. 08/311,61 1 filed Sep. 22, 1994 and WO95/08344 (PCT/US94/11225). Specifically, concurrent administration, or co-treatment, of such BPI protein products and an antibiotic or combination of antibiotics may improve the therapeutic effectiveness of antibiotics in a variety of ways, including by increasing susceptibility of gram-negative bacteria to a reduced dosage of antibiotics, by effectively reversing resistance of gram-negative bacteria to antibiotics, by providing synergistic or potentiating effects beyond the individual or additive effects of the BPI protein product or antibiotic alone, or by neutralizing endotoxin released by bacteria killed by antibiotics. Concurrent administration of BPI protein products and antibiotics provide unexpectedly superior therapeutic effects in vivo than either agent provides when administered alone. In particular, concurrent administration of BPI protein product according to this improved method of treatment is effective even when the gram-negative bacteria involved are considered to be resistant to the bactericidal effects of BPI protein product alone and/or antibiotic alone. BPI protein products are therefore useful for prophylaxis or treatment of gram-negative bacterial infections, including for prophylaxis of patients at high risk of gram-negative bacterial infection, e.g., patients who will undergo abdominal or genitourinary surgery, or trauma victims.  
       [0010] BPI protein products, including BPI-derived peptides, have been shown recently to have direct and indirect bactericidal and growth inhibitory effects on some gram-positive organisms as described in copending U.S. patent application Ser. No. 08/372,783 filed Jan. 13, 1995 and WO95/19180 (PCT/US95/00656). In addition, BPI protein products unexpectedly were shown to have the ability to increase the antibiotic susceptibility of gram-positive bacteria, including the ability to reverse in many instances the antibiotic resistance of gram-positive bacteria. BPI protein products and antibiotics provided additive and synergistic bactericidal/growth inhibitory effects when administered concurrently. Such BPI protein products are therefore useful for treating gram-positive bacterial infections, including conditions associated therewith or resulting therefrom (for example, sepsis or bacteremia).  
       [0011] BPI protein products, including BPI-derived peptides, have also been shown recently to have fungicidal/fungistatic effects as described in copending and co-assigned U.S. patent application Ser. No. 08/372,105 filed Jan. 13, 1995 and WO95/19179 (PCT/US95/00498). Such BPI protein products may be administered alone or in conjunction with known anti-fungal agents. When made the subject of adjunctive therapy, the administration of BPI protein products may reduce the amount of anti-fungal agent needed for effective therapy, thus limiting potential toxic response and/or high cost of treatment. Administration of BPI protein products may also enhance the effect of such agents, accelerate the effect of such agents, or reverse resistance of fungi to such agents.  
       [0012] BPI has other important biological activities. For example, BPI protein products, including BPI-derived peptides, have been shown to have heparin binding and heparin neutralization activities in copending and co-assigned U.S. Pat. No. 5,348,942 issued Sep. 20, 1994 incorporated by reference herein. These heparin binding and neutralization activities are significant due to the importance of current clinical uses of heparin. Heparin is commonly administered in doses of up to 400 U/kg during surgical procedures such as cardiopulmonary bypass, cardiac catherization and hemodialysis procedures in order to prevent blood coagulation during such procedures. When heparin is administered for anticoagulant effects during surgery, it is an important aspect of post-surgical therapy that the effects of heparin are promptly neutralized so that normal coagulation function can be restored. Currently, protamine is used to neutralize heparin. Protamines are a class of simple, arginine-rich, strongly basic, low molecular weight proteins. Administered alone, protamines (usually in the form of protamine sulfate) have anti-coagulant effects. When administered in the presence of heparin, a stable complex is formed and the anticoagulant activity of both drugs is lost. However, significant hypotensive and anaphylactoid effects of protamine have limited its clinical utility. Thus, due to its heparin binding and neutralization activities, BPI has potential utility as a substitute for protamine in heparin neutralization in a clinical context without the deleterious side-effects which have limited the usefulness of the protamines. The additional antibacterial and anti-endotoxin effects of BPI would also be useful and advantageous in post-surgical heparin neutralization compared with protamine.  
       [0013] Additionally, BPI protein products are useful in inhibiting angiogenesis due in part to its heparin binding and neutralization activities. In adults, angiogenic growth factors are released as a result of vascular trauma (wound healing), immune stimuli (autoimmune disease), inflammatory mediators (prostaglandins) or from tumor cells. These factors induce proliferation of endothelial cells (which is necessary for angiogenesis) via a heparin-dependent receptor binding mechanism. Angiogenesis is also associated with a number of other pathological conditions, including the growth, proliferation, and metastasis of various tumors; diabetic retinopathy, retrolental fibroplasia, neovascular glaucoma, psoriasis, angiofibromas, immune and non-immune inflammation including rheumatoid arthritis, capillary proliferation within atherosclerotic plaques, hemangiomas, endometriosis and Kaposi&#39;s sarcoma. Thus, it would be desirable to inhibit angiogenesis in these and other instances, and the heparin binding and neutralization activities of BPI are useful to that end.  
       [0014] Another utility of BPI protein products involves pathological conditions associated with chronic inflammation, which is usually accompanied by angiogenesis. One example of a human disease related to chronic inflammation is arthritis, which involves inflammation of peripheral joints. In rheumatoid arthritis, the inflammation is immune-driven, while in reactive arthritis, inflammation is associated with infection of the synovial tissue with pyogenic bacteria or other infectious agents. Many types of arthritis progress from a stage dominated by an inflammatory infiltrate in the joint to a later stage in which a neovascular pannus invades the joint and begins to destroy cartilage. While it is unclear whether angiogenesis in arthritis is a causative component of the disease or an epiphenomenon, there is evidence that angiogenesis is necessary for the maintenance of synovitis in rheumatoid arthritis. BPI has been shown to provide effective therapy for arthritis and other inflammatory diseases.  
       [0015] Three separate functional domains within the recombinant 23 kD N-terminal BPI sequence were discovered by Little et al.,  J. Biol. Chem.  269: 1865 (1994) [see also copending and co-assigned WO94/20128 (PCT/US94/02401); WO94/20532 (PCT/US94/02465);and WO95/19372 (PCT/US94/10427)]. These functional domains of BPI designate a region of the amino acid sequence of BPI that contributes to the total biological activity of the protein and were essentially defined by the activities of proteolytic cleavage fragments, overlapping 15-mer peptides and other synthetic peptides. Domain I is defined as the amino acid sequence of BPI comprising from about amino acid 17 to about amino acid 45. Peptides derived from this domain were moderately active in both the inhibition of LPS-induced LAL activity and in heparin binding assays, and did not exhibit significant bactericidal activity. Domain II is defined as the amino acid sequence of BPI comprising from about amino acid 65 to about amino acid 99. Peptides derived from or based on this domain exhibited high LPS and heparin binding capacity and were bactericidal. Domain m is defined as the amino acid sequence of BPI comprising from about amino acid 142 to about amino acid 169. Peptides derived from or based on this domain exhibited high LPS and heparin binding activity and were bactericidal. The biological activities of BPI functional domain peptides may include LPS binding, LPS neutralization, heparin binding, heparin neutralization or antimicrobial activity.  
       [0016] Of interest to the present application are the disclosures of the following references which relate to recombinant fusion proteins and peptides.  
       [0017] Shen,  Proc. Nat&#39;l. Acad. Sci.  ( USA ), 281:4627 (1984) describes bacterial expression as insoluble inclusion bodies of a fusion protein encoding pro-insulin and β-galactosidase; the inclusion bodies were solubilized with formic acid prior to cleavage with cyanogen bromide.  
       [0018] Kempe et al.,  Gene,  39:239 (1985) describes expression as insoluble inclusion bodies in  E. coli  of a fusion protein encoding multiple units of neuropeptide substance P and β-galactosidase; the inclusion bodies were solubilized with formic acid prior to cleavage with cyanogen bromide.  
       [0019] Lennick et al.,  Gene,  61:103 (1987) describes expression as insoluble inclusion bodies in  E. coli  of a fusion protein encoding multiple units (8) of α-human atrial natriuretic peptide; the inclusion bodies were solubilized with urea prior endoproteinase cleavage.  
       [0020] Dykes et al.,  Eur. J. Biochem.,  174.411 (1988) describes soluble intracellular expression in  E. coli  of a fusion protein encoding α-human atrial natriuretic peptide and chloramphenicol acetyltransferase; the fusion protein was proteolytically cleaved or chemically cleaved with 2-(2-nitrophenylsulphenyl)-E-methyl-3′-bromoindolenine to release peptide.  
       [0021] Ray et al.,  Bio/Technology,  11:64 (1993) describes soluble intracellular expression in  E. coli  of a fusion protein encoding salmon calcitonin and glutathione-S-transferase; the fusion protein was cleaved with cyanogen bromide.  
       [0022] Schellenberger et al.,  Int. J. Peptide Protein Res.,  41:326 (1993) describes expression as insoluble inclusion bodies of a fusion protein encoding a substance P peptide (11a.a.) and β-galactosidase; the inclusion bodies were treated with chymotrypsin to cleave the fusion protein.  
       [0023] Hancock et al., WO94/04688 (PCT/CA93/00342) and Piers et al. (Hancock),  Gene,  134:7 (1993) describe (a) expression as insoluble inclusion bodies in  E. coli  of a fusion protein encoding a defensin peptide designated human neutrophil peptide 1 (HNP-1) or a hybrid cecropin/mellitin (CEME) peptide and glutathione-5-transferase (GST); the inclusion bodies were: (i) extracted with 3% octyl-polyoxyethylene prior to urea solubilization and prior to factor X a  protease for HNP1-GST fusion protein or (ii) solubilized with formic acid prior to cyanogen bromide cleavage for CEME-GST fusion protein; (b) expression in the extracellular supernatant of  S. aureus  of a fusion protein encoding CEME peptide and protein A; (c) proteolytic degradation of certain fusion proteins with some fusion protein purified; and (d) proteolytic degradation of other fusion proteins and inability to recover and purify the fusion protein.  
       [0024] Lai et al., U.S. Pat. No. 5,206.154 and Callaway, Lai et al.  Antimicrob. Agents  &amp;  Chemo.,  37:1614 (1993) describe expression as insoluble inclusion bodies of a fusion protein encoding a cecropin peptide and the protein encoded by the 5′-end of the L-ribulokinase gene; the inclusion bodies were solubilized with formic acid prior to cleavage with cyanogen bromide.  
       [0025] Gamm etal.,  Bio/Technology,  12:1017 (1994) describes expression as insoluble inclusion bodies in  E. coli  of a fusion protein encoding a human parathyroid hormone peptide and a bacteriophage T4-encoded gp55 protein; the inclusion bodies (6% wt/vol.) were treated with acid to hydrolyze the Asp-Pro cleavage site.  
       [0026] Kuliopulos et al.,  J. Am. Chem. Soc.,  116:4599 (1994) describes expression as insoluble inclusion bodies in  E. coli  of a fusion protein encoding multiple units of a yeast a-mating type peptide and a bacterial ketosteroid isomerase protein; the inclusion bodies were solubilized with guanidine prior to cyanogen bromide cleavage.  
       [0027] The above-references indicate that production of small peptides from bacteria has been problematic for a variety of reasons. Proteolysis of some peptides has been particularly problematic, even where the peptide is made as a part of a larger fusion protein. Such fusion proteins comprising a carrier protein/peptide may not be expressed by bacterial host cells or may be expressed but cleaved by bacterial proteases. In particular, difficulties in expressing cationic antimicrobial peptides in bacteria have been described by Hancock et al. WO94/04688 (PCT/CA93/00342) referenced above, due in their view to the susceptibility of such polycationic peptides to bacterial protease degradation.  
       [0028] There continues to exist a need in the art for new recombinant products and in particular, a need for methods for recombinant production of BPI-derived peptides useful as antimicrobial agents (including anti-bacterial and anti-fungal agents), as endotoxin binding and neutralizing agents, and as heparin binding and neutralizing agents, including agents for neutralizing the anticoagulant effects of administered heparin, for treatment of chronic inflammatory disease states, and for inhibition of normal or pathological angiogenesis.  
       SUMMARY OF THE INVENTION  
       [0029] The present invention provides methods and materials for recombinant microbial production of fusion proteins and peptides derived from or based on bactericidal/permeability-increasing protein (BPI). Preferred BPI peptides are derived from Domain I (amino acids 17-45), Domain II (amino acids 65-99) and Domain m (amino acids 142-169) of BPI, each peptide having an amino acid sequence that is the amino acid sequence of a BPI functional domain or a subsequence thereof and variants of the sequence or subsequence having at least one of the biological activities of BPI. Fusion proteins of the invention comprise at least one BPI peptide sequence, a carrier protein sequence, and at least one amino acid cleavage site sequence located between the BPI peptide and the carrier protein sequence. The invention provides a method for the microbial production of such fusion proteins encoding one or more BPI peptides. The recombinant BPI-derived peptides of the invention are released by cleavage at the cleavage site(s) in the fusion protein. Such peptides are efficiently and economically produced according to the invention.  
       [0030] Methods of the invention for recombinant microbial production of fusion proteins and BPI-derived peptides are based on the surprising discovery that such fusion proteins are expressed in large amounts intracellularly or secreted from microbial host cells, that antimicrobial BPI peptides are efficiently produced by microbial host cells and that the peptides are efficiently cleaved and released from the fusion proteins. It is particularly surprising that antibacterial BPI peptides according to the invention are effectively made in  E. coli . Such BPI-derived peptides having one or more of the biological activities of BPI can be isolated and purified according to the invention. Thus, the invention provides functional recombinant BPI peptides. Biologically active recombinant BPI peptides of the invention released from the fusion proteins have one or more of the following activities: LPS binding, LPS neutralization, heparin binding, heparin neutralization or antimicrobial activity (including anti-bacterial, anti-fungal activity).  
       [0031] As such, recombinant BPI-derived peptides are useful as antimicrobial agents (including anti-bacterial and anti-fungal agents), as endotoxin binding and neutralizing agents, and as heparin binding and neutralizing agents including agents for neutralizing the anticoagulant effects of administered heparin, for treatment of chronic inflammatory disease states, and for inhibition of normal or pathological angiogenesis.  
       [0032] The invention provides recombinant DNA vector constructs suitable for introduction into a bacterial host in which the construct includes a coding sequence for a fusion protein having: (a) at least one cationic BPI peptide encoding DNA sequence; (b) a carrier protein encoding DNA sequence; and (c) an amino acid cleavage site encoding DNA sequence located between the sequences (a) and (b). According to the invention, a preferred vector construct is provided with a coding sequence for a fusion protein is 5′-(b)-(c)-(a)-3′, that is, from 5′ to 3′, a carrier encoding sequence, followed by a cleavage site encoding sequence, and then a peptide encoding sequence. The invention further provides an encoded BPI peptide that is bactericidal, fungicidal, endotoxin binding, endotoxin neutralizing, heparin binding or heparin neutralizing. According to one aspect of the invention, an encoded BPI peptide is provided that comprises an amino acid sequence of SEQ ID NOS: 1-239. The invention also provides an encoded carrier protein that is a cationic carrier protein, for example, gelonin or the D subunit of human osteogenic protein. Constructs are also provided that additionally encode a bacterial secretory leader sequence at the amino-terminus of the fusion protein. An encoded amino acid cleavage site is provided in vector constructs, including codons encoding Asp-Pro, Met, Trp and Glu. Bacterial host cells transformed with vector constructs according to the invention are provided, including  E. coli  host cells.  
       [0033] The invention provides methods for bacterial production of fusion proteins and BPI peptides by culturing transformed bacterial host cells transformed with a vector construct encoding the fusion protein that has BPI peptide-, carrier protein-, and amino acid cleavage site—encoding sequences, by optionally isolating the expressed fusion protein, by cleaving the expressed fusion protein to release the BPI peptide, and by isolating the BPI peptide. BPI peptide products of processes according to the invention are provided. Additionally, methods for bacterial production of fusion proteins are provided, which includes culturing a bacterial host cell transformed with a vector construct encoding the fusion protein, and isolating the expressed fusion protein. Fusion protein products of such processes are also provided. 
     
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
     [0034]FIG. 1 shows the acid liability of the Asp-Pro peptide linker over time at various temperatures.  
     [0035]FIG. 2 shows an Arrhenius plot for the hydrolysis of fusion protein.  
     [0036]FIG. 3 shows acid cleavage rates of various acid-treated inclusion body suspensions.  
     [0037]FIG. 4 shows maximal peptide release achieved with 60 mM and 90 mM HCl treated suspensions.  
     [0038]FIG. 5 shows the highest yield of product from pING3360 with four peptide repeat units. 
    
    
     DETAILED DESCRIPTION  
     [0039] The present invention provides recombinant methods and compositions of fusion proteins and BPI peptides encoded by and released from such fusion proteins. Unexpectedly, such fusion proteins containing BPI-derived peptides with anti-microbial activity can be expressed in large amounts without significant proteolysis, and in some cases are actually secreted from microbial host cells. A variety of BPI-derived peptides, including those comprising the sequences listed in Table 4 (SEQ ID NOS: 1-239), may be produced by recombinant methods according to the invention. Such BPI-derived peptides having at least one or the activities of BPI (e.g., LPS binding, LPS neutralization, heparin binding, heparin neutralization or antimicrobial activity (including anti-bacterial, anti-fungal) may be useful as antimicrobial agents (including anti-bacterial and anti-fungal agents), as endotoxin binding and neutralizing agents, and as heparin binding and neutralizing agents including agents for neutralizing the anticoagulant effects of administered heparin, for treatment of chronic inflammatory disease states, and for inhibition of normal or pathological angiogenesis.  
     [0040] An advantage provided by present invention is the ability to produce efficiently and economically from bacterial host cells such BPI peptides. Additional advantages include the ability to obtain homogeneous peptide in large amounts via methods that are amenable to scale-up.  
     [0041] “BPI-derived peptide” or “BPI peptide” as used herein refers to a peptide derived from or based on bactericidal/permeability-increasing protein (BPI), including peptides derived from Domain I (amino acids 17-45), Domain II (amino acids 65-99) and Domain III (amino acids 142-169) of BPI, each peptide having an amino acid sequence that is the amino acid sequence of a BPI functional domain or a subsequence thereof and variants of the sequence or subsequence having at least one of the biological activities of BPI. As used herein, a “biological activity of BPI” refers to LPS binding, LPS neutralization, heparin binding, heparin neutralization or antimicrobial activity (including anti-bacterial and anti-fungal activity. As used herein, “cationic BPI peptide” refers to a BPI peptide with a pI&gt;7.0, as exemplified by the peptides listed in Table 4 herein.  
     [0042] As used herein a “transformed bacterial host cell refers to a bacterial cell that contains recombinant genetic material or a bacterial cell that contains genetic material required for expression of a recombinant product. The genetic material may be introduced by any method known in the art including transformation, transduction, eletroporation and infection.  
     [0043] As used herein, a “vector construct” refers to plasmid DNA that contains recombinant genetic material which may encode a recombinant product(s) and may be capable of autonomous replication in bacteria.  
     [0044] “Carrier protein” as used herein refers to a protein that can be expressed in bacteria and used as a fusion partner to a linked peptide or protein. Preferred carrier proteins are those that can be expressed at high yield and when used as a fusion partner can confer high level-expression to a linked peptide or protein. A “cationic carrier protein” as used herein refers to a carrier protein having a pI (as calculated based on amino acid sequence or as measured in solution) greater than 7.0 and preferably greater than 8.0. Such preferred proteins include gelonin (pI 9.58) and the D subunit of human osteogenic protein (pI 8.18).  
     [0045] “Amino acid cleavage site” as used herein refers to an amino acid or amino acids that serve as a recognition site for a chemical or enzymatic reaction such that the peptide chain is cleaved at that site by the chemical agent or enzyme. Preferred amino acid cleavage sites are at aspartic acid—proline (Asp-Pro), methionine (Met), tryptophan (Trp) or glutamic acid (Glu). Particularly preferred is the Asp-Pro cleavage site which may be cleaved between Asp and Pro by acid hydrolysis.  
     [0046] As used herein, “BPI protein product” includes naturally and recombinantly produced BPI protein: natural, synthetic, and recombinant biologically active polypeptide fragments of BPI protein; biologically active polypeptide variants of BPI protein or fragments thereof, including hybrid fusion proteins and dimers; biologically active polypeptide analogs of BPI protein or fragments or variants thereof, including cysteine-substituted analogs; and BPI-derived peptides. The BPI protein products administered according to this invention may be generated and/or isolated by any means known in the art. U.S. Pat. No. 5,198,541, the disclosure of which is incorporated herein by reference, discloses recombinant genes encoding and methods for expression of BPI proteins including recombinant BPI holoprotein, referred to as rBPI 50  and recombinant fragments of BPI. Co-owned, copending U.S. patent application Ser. No. 07/885,501 and a continuation-in-part thereof, U.S. patent application Ser. No. 08/072,063 filed May 19, 1993 and corresponding PCT Application No. 93/04752 filed May 19, 1993, which are all incorporated herein by reference, disclose novel methods for the purification of recombinant BPI protein products expressed in and secreted from genetically transformed mammalian host cells in culture and discloses how one may produce large quantities of recombinant BPI products suitable for incorporation into stable, homogeneous pharmaceutical preparations.  
     [0047] Biologically active fragments of BPI (BPI fragments) include biologically active molecules that have the same or similar amino acid sequence as a natural human BPI holoprotein, except that the fragment molecule lacks amino-terminal amino acids, internal amino acids, and/or carboxy-terminal amino acids of the holoprotein. Nonlimiting examples of such fragments include a N-terminal fragment of natural human BPI of approximately 25 kD, described in Ooi et al.,  J. Exp. Med.,  174:649 (1991), and the recombinant expression product of DNA encoding N-terminal amino acids from 1 to about 193 or 199 of natural human BPI, described in Gazzano-Santoro et al.,  Infect. Immun.  60:4754-4761 (1992), and referred to as rBPI 23 . In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI 23 ) having the 31-residue signal sequence and the first 199 amino acids of the N-terminus of the mature human BPI. as set out in FIG. 1 of Gray et al., supra, except that valine at position 151 is specified by GTG rather than GTC and residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG). Recombinant holoprotein (rBPI) has also been produced having the sequence (SEQ ID NOS: 145 and 146) set out in FIG. 1 of Gray et al., supra, with the exceptions noted for rBPI 23  and with the exception that residue 417 is alanine (specified by GCT) rather than valine (specified by GT). Other examples include dimeric forms of BPI fragments, as described in co-owned and co-pending U.S. patent application Ser. No. 08/212,132, filed Mar. 11, 1994, and corresponding PCT Application No. 95/03125, the disclosures of which are incorporated herein by reference. Preferred dimeric products include dimeric BPI protein products wherein the monomers are amino-terminal BPI fragments having the N-terminal residues from about 1 to 175 to about 1 to 199 of BPI holoprotein. A particularly preferred dimeric product is the dimeric form of the BPI fragment having N-terminal residues 1 through 193, designated rBPI 42  dimer.  
     [0048] Biologically active variants of BPI (BPI variants) include but are not limited to recombinant hybrid fusion proteins, comprising BPI holoprotein or biologically active fragment thereof and at least a portion of at least one other polypeptide, or dimeric forms of BPI variants. Examples of such hybrid fusion proteins and dimeric forms are described by Theofan et al. in co-owned, copending U.S. patent application Ser. No. 07/885,911, and a continuation-in-part application thereof, U.S. patent application Ser. No. 08/064,693 filed May 19, 1993 and corresponding PCT Application No. US93/04754 filed May 19, 1993, which are all incorporated herein by reference and include hybrid fusion proteins comprising, at the amino-terminal end, a BPI protein or a biologically active fragment thereof and, at the carboxy-terminal end, at least one constant domain of an immunoglobulin heavy chain or allelic variant thereof. Another example of such a hybrid fusion protein is the recombinant expression product of DNA encoding amino acids 1 through 199 of BPI joined to DNA encoding amino acids 198 through 456 of LBP, designated BPI(1-199)-LBP(198-456) hybrid, is described in PCT Application No. US94/06931 filed Jun. 17, 1994, which corresponds to co-owned, co-pending U.S. patent application Ser. No. 08/261,660, filed Jun. 17, 1994 as a continuation-in-part of U.S. patent application Ser. No. 08/079,510, filed Jun. 17, 1993, the disclosures of all of which are incorporated herein by reference.  
     [0049] Biologically active analogs of BPI (BPI analogs) include but are not limited to BPI protein products wherein one or more amino acid residues have been replaced by a different amino acid. For example, co-owned, copending U.S. patent application Ser. No. 08/013,801 filed Feb. 2, 1993 and corresponding PCT Application No. US94/01235 filed Feb. 2, 1994, the disclosures of which are incorporated herein by reference, discloses polypeptide analogs of BPI and BPI fragments wherein a cysteine residue is replaced by a different amino acid. A preferred BPI protein product described by this application is the expression product of DNA encoding from amino acid 1 to approximately 193 or 199 of the N-terminal amino acids of BPI holoprotein, but wherein the cysteine at residue number 132 is substituted with alanine and is designated rBPI 21 Δcys or rBPI 21 . Other examples include dimeric forms of BPI analogs; e.g. co-owned and co-pending U.S. patent application Ser. No. 08/212,132 filed Mar. 11, 1994, and corresponding PCT Application No. 95/03125, the disclosures of which are incorporated herein by reference.  
     [0050] BPI protein products also include peptides derived from or based on BPI (BPI-derived peptides), such as those described in co-owned and copending U.S. patent application Ser. No. 08/621,259 entitled “Anti-Fungal Peptides” filed Mar. 21, 1996 (Attorney Docket No. 27129/33198); PCT/US95/09262 and U.S. patent application Ser. No. 08/504,841 filed Jul. 20, 1995; WO95/19372 (PCT Application No. US94/10427) and U.S. patent application Ser. No. 08/306,473 filed Sep. 15, 1994; WO94/20532 (PCT Application No. US94/02465) and U.S. patent application Ser. No. 08/209,762, filed Mar. 11, 1994; WO94/20128 (PCT Application No. US94/02401) and U.S. patent application Ser. No. 08/093,202 filed Jul. 15, 1993, the disclosures of all of which are incorporated herein by reference.  
     [0051] Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples wherein Example 1 addresses construction of fusion protein expression vector constructs; Example 2 addresses expression of recombinant fusion proteins; Example 3 addresses isolation of inclusion bodies from cells expressing intracellular recombinant product; Example 4 addresses Isolation of secreted fusion protein and purification of recombinant peptide from bacterial host cell culture media; Example 5 addresses radial diffusion assays for antimicrobial activity analysis of recombinant peptides; and Example 6 addresses additional biological activity assays of recombinant peptides.  
     EXAMPLE 1  
     Construction of Fusion Protein Expression Vector Constructs  
     [0052] 1. Bacterial Expression Vector Construct: pING3793  
     [0053] A bacterial expression vector which would encode a peptide fusion protein, was constructed. This vector contains a sequence for a gene encoding gelonin (see, amino acids 23 through 273 of SEQ ID NOS: 250 and 251) linked to a sequence encoding an SLT linker (see, amino acids 277 through 296 of SEQ ID NOS: 250 and 251) and a sequence encoding a peptide derived from BPI comprising amino acids corresponding to 85-99 and 148-162 of BPI (SEQ ID NO: 265). A unique BamIHi site was incorporated into this vector at the junction of gelonin and peptide encoding DNA along with an encoded Asp-Pro dipeptide. For the experiments described herein, restriction and modification enzymes were purchased from New England Biolabs, Beverley, Mass. and GIBCO/BRL, Gathersberg, Md. This expression vector, pING3793, was constructed from a previously described gelonin containing vector designated pING3748 and described in U.S. Pat. No. 5,416,202 incorporated by reference in its entirety (see, e.g., Examples 10 and 2). Plasmid pING3748, which contains a gelonin gene linked to SLT, was cut with Scal and XhoI. The vector fragment was ligated to annealed and extended oligonucleotides that encoded the BPI-derived peptide. The oligonucleotides encoding the BPI-derived peptide were:  
     [0054] 5′-GCTATCTGCGCATTGGATCCGATCAAAATCTCGGGTAAATGGAAGGC CCAGAAACGCTTTCTGAAAAAGTCGAAAGTG-3′ SEQ ID NO: 240  
     [0055] and  
     [0056] 5′-GCGGGCTCTCGAGCTTTAAATCTTTATGAAACAGCTGGATCAGCCA ACCCACTTTCGACTTTTTCAGAAAGCG-3′ SEQ ID NO: 241  
     [0057] 16  μg of each oligo were annealed in 10 mM TRIS, pH 8, 100 mM NaCl, 0.1 mM EDTA. Annealed oligos were extended with AmpliTaq® (Perkin Elmer, Norwalk, Conn.) in a 50 μL reaction containing standard PCR reagents in a Gene Amps kit (Perkin Elmer, Norwalk, Conn.) according to the manufacturer&#39;s instructions) for 10 minutes at 72° C. The extended DNA fragment was purified on a Chroma-spin 30 column (Clonetech, Palo Alto, Calif.) and digested with FspI and XhoI. The purified DNA fragment was ligated to the pING3748 vector fragment. The ligated DNA was used to transform  E. coli  MC 1061. A candidate clone containing the DNA insert was identified by restriction analysis. The sequence of the fusion protein encoded by pING3793 is shown in SEQ ID NOS: 250 and 251.  
     [0058] 2. Bacterial Expression Vector Construct: pING3795  
     [0059] An expression vector construct was prepared from pING3793 that contained a gelonin gene (see amino acids 23 through 273 of SEQ ID NOS: 250 and 251) linked to a BPI-derived peptide comprising amino acids 85-99 and 148-162 of BPI (SEQ ID NO: 265) but that lacked the SLT region described above. To accomplish this, DNA segments from three plasmids were cloned together. Plasmid pING3825 described in U.S. Pat. No. 5,416,202, incorporated by reference (see, e.g., Example 2). which encodes a recombinant gelonin, was digested with NcoI and HindIII. The vector fragment containing the 5′-end of this gelonin gene was purified. Plasmid pING3755 described in U.S. Pat. No. 5,416,202 (see, e.g., Example 10) incorporated by reference, was cut with EagI, treated with T4 polymerase to fill in the 5′-overhang, and digested with NcoI. The approximately 650 bp DNA fragment was purified. Plasmid 3793 was digested with FspI and HindIII, and the approximately 175 bp DNA fragment was purified. The three isolated DNA fragments were ligated to generate pING3795. The ligated DNA was used to transform  E. coli  MC 1061. A candidate clone containing the correct DNA insert was identified by restriction analysis and the DNA sequence of the candidate clone, pING3793, was verified by sequencing with Sequenase™ (US Biochemical, Cleveland, Ohio). A candidate clone containing the correct DNA insert was identified by restriction analysis. The sequence of the fusion protein encoded by pING3795 is shown in SEQ ID NOS: 252 and 253.  
     [0060] 3. Bacterial Expression Vector Construct: pING3353  
     [0061] The DNA segment encoding a BPI-derived peptide comprising amino acids 85-99 and 148-162 of BPI (SEQ ID NO: 265) was cloned onto the 3′-end of a gene encoding subunit D of a human osteogenic protein (“Bone D”) protein vector (see, amino acids 23 through 161 of SEQ ID NOS: 248 and 249) to prepare a vector construct encoding a peptide fusion protein. This vector construct, pING3353, was prepared as described below and the vector encodes a Bone D protein, a Asp-Pro dipeptide, a BPI-derived peptide segment, and contains the unique BamHI restriction site (See, SEQ ID NOS: 254 and 255).  
     [0062] The Bone D gene described above linked to a pel B leader sequence (see amino acids 1 through 161 of SEQ ID NOS: 248 and 249), as resident on plasmid pING3913, contains a BfaI restriction site at the 3′-end of the coding region. Plasmid pING3913 encodes a gene encoding subunit D of human osteogenic protein that is described in U.S. Pat. No. 5,284,756 incorporated by reference in its entirety (see Example 9; FIG. 6 and SEQ ID NO: 2). Digestion of pING3913 with BfaI followed by treatment of the 5′-overhang with mung bean nuclease generates a blunt end which encodes up to and including the last amino acid of the Bone D gene, which is amino acid 139 (histidine). pING3913 was cut with BfaI, treated with mung bean nuclease and then cut with EcoRI. The approximately 550 bp DNA fragment encoding Bone D was then purified. Plasmid pING3793 (see Section 1 above) was cut with FspI and HindIII and the DNA segment containing the BamHI site and encoding an Asp-Pro dipeptide and a BPI-derived peptide (approximately 175 bp) was purified. These two DNA fragments were cloned into an  E. coli  expression vector containing an Ara B expression system (e.g., pING3737/ATCC 69009; pING3746/ATCC 69008; pING3747/ATCC 69101; pING3754/ATCC 69102; pING3758/ATCC69103; pING3759/ATCC 69104; pING3336/ATCC69331; pING4644/ATCC 69332; pING4629/ATCC 69333); see U.S. Pat. No. 5,416,202) which had been digested with EcoRI and HindIII to generate pING3353. The ligated DNA was used to transform  E. coli  MC1061. A candidate clone containing the correct DNA insert was identified by restriction analysis. The DNA sequence at the junction of the Bone D gene and the peptide segment of pING3353 was verified by DNA sequencing with Sequenase™ (US Biochemical, Cleveland, Ohio).  
     [0063] 4. Intermediate Vector Construct: pING3354  
     [0064] DNA encoding four BPI-derived peptides were cloned into a plasmid vector as fusions to the Bone D gene. Degenerate oligonucleotides were synthesized which could encode these peptides. These oligonucleotides were degenerate at two positions and could encode four possible peptides with amino acids F, A, S and V at the position corresponding to residue 153 in BPI.  
     [0065] The two oligos:  
     [0066] 5′-GATC CGAAGTCTAAAGTGGGG[G/T][G/T]CCTGATCCAGCTGTTCCACA AAAAGTAAAGC-3′ SEQ ID NO: 242  
     [0067] and  
     [0068] 5′-TCGAGTCTTACTTTTTGTGAAGCAGCTGGATCAGG[G/A][C/A]CCCCAC TTTAGACTTCG-3′ SEQ ID NO: 243  
     [0069] were synthesized and purified on a 10% acrylamide gel. Approximately 1 μg of each was annealed in 10 mM TRIS, pH 8, 100 mM NaCl, 0.1 mM EDTA. Plasmid pING3353 (see Section 3 above) was cut with EcoRI and BamHI, and the approximately 550 bp fragment containing the Bone D gene was purified. The plasmid pIC100, a derivative of pBR322, and which includes the leader sequence of the E. carotovora pel B gene, described in U.S. Pat. No. 5,416,202 (see, e.g., Example 10) incorporated by reference, was cut with EcoRI and XhoI and the vector fragment was purified. The annealed oligos were ligated to the digested pING3353 and pIC100 to generate four plasmids containing cloned peptide fusions. The ligated DNA was used to transform  E. coli  MC1061. Candidate clones containing a DNA insert were identified by restriction analysis. The plasmid encoding a peptide fusion protein with alanine at the position in the peptide corresponding to residue 153 in BPI was designated pING3354. The plasmid encoding a peptide fusion protein with serine at the position in the peptide corresponding to residue 153 of BPI was designated pING3355. The plasmid encoding a peptide fusion protein with valine at the position in the peptide corresponding to residue 153 in BPI was designated pING3356. The plasmid encoding a peptide fusion protein with phenylalanine at the position in the peptide corresponding to residue 153 in BPI was designated pING3357. Plasmid pING3354 was distinguished by restriction analysis by a unique Apal site at the position of the encoded alanine. The DNA sequences of all four peptide-encoding plasmids were verified by DNA sequence determination with Sequenase™ (US Biochemical, Cleveland, Ohio). The sequence of the fusion protein encoded by plasmid pING3354 is shown in SEQ ID NOS: 256 and 257. The sequences of the fusion proteins encoded by pING3355, pING3356, and pING3357 are identical to the fusion protein shown in SEQ ID NO: 257 except that the residue corresponding to 153 in BPI is serine, valine and phenylalanine, respectively.  
     [0070] 5. Bacterial Expression Vector Construct: pING3797  
     [0071] The DNA encoding the sequence of a BPI-derived antifungal peptide previously designated as XMP.36 from intermediate vector pING3354 was cloned onto the 3′-end of a gelonin gene. The resultant plasmid, pING3797, which encodes the Asp-Pro dipeptide between the gelonin and peptide gene segments was prepared as follows.  
     [0072] Plasmid pING3825, which encodes recombinant gelonin, was digested with NcoI and XhoI, and the vector fragment containing the 5′-end of the gelonin gene was purified. Plasmid pING3795 was digested with NcoI and BamHI, and the approximately 650 bp fragment containing the 3′-end of the gelonin gene as purified. Plasmid pING3354 was cut with BamlE and XhoI. These three DNA fragments were ligated together to generate pING3797 encoding the gelonin peptide fusion protein. The ligated DNA was used to transform  E. coli  MC1061. Candidate clones containing the correct DNA inserts were identified by restriction analysis. The sequence of the fusion protein encoded by plasmid pING3797 is shown in SEQ ID NOS: 258 and 259.  
     [0073] 6. Bacterial Expression Vector Construct: pING3796  
     [0074] The Bone D gene fused to the DNA encoding the BPI-derived antifungal peptide from intermediate vector pING3354 was cloned into a bacterial expression vector. Plasmid pING3354 was cut with EcoRI and XhoI and the approximately 610 bp DNA fragment encoding the entire Bone D and peptide sequences was purified. This DNA fragment was cloned into the vector fragment of pING3217 that had been cut with EcoRi and XhoI and purified. This vector fragment is identical to the vector fragment obtained from pING3737/ATCC 69009 (or alternatively, pING3746/ATCC 69008; pING3747/ATCC 69101; pING3754/ATCC 69102;; pING3758/ATCC69103; pING3759/ATCC 69104; pING3336/ATCC69331; pING4644/ATCC 69332; and pING4629/ATCC 69333) cut with EcoRI and XhoI. The ligated DNA was used to transform  E. coli  MC1061. Candidate clones containing the correct DNA inserts were identified by restriction analysis. The resultant plasmid was pING3796. The sequence of the fusion protein encoded by plasmid pING 3796 is shown in SEQ ID NOS: 260 and 261.  
     [0075] 7. Bacterial Expression Vector Constructs Encoding Bone D Fusions and Repeat Units of a BPI-derived Peptide: pING3359, pING3360, pING3361, and pING3362  
     [0076] Plasmid pING3796 encodes a single peptide segment linked to the 3′-end of Bone D with an encoded Asp-Pro dipeptide in between (see SEQ ID NOS: 260 and 261). Several similar expression vectors were constructed that contained repeat units of this peptide segment separated by Asp-Pro encoding segments.  
     [0077] To construct the repeat units, two oligonucleotide primers were synthesized and used to amplify the Bone D and peptide-encoding DNA sequences. The resulting PCR product was cut with ApaI, and a 48 bp peptide encoding unit was purified. This DNA fragment was self ligated under conditions where the repeat units containing 2, 3, 4, and 5 peptide-encoding segments were the predominant products. These Ligation products were purified on an agarose gel and ligated into the unique ApaI site present in pING3354 at the position encoding the amino acid corresponding to residue 153 in BPI. Candidate clones containing 2-5 repeats units of the desired peptide segment were identified by restriction analysis, and their DNA sequences were verified directly with Sequenase™ (US Biochemicals, Cleveland, Ohio). The repeat containing segments were then cloned into a bacterial expression vector in a manner analogous to that described above for the construction of pING3796 from pING3354.  
     [0078] Specifically, two oligonucleotide primers were synthesized:  
     [0079] 5′-ACTTGGGCCCCTACCTTGGATTTTGGGTCCTTTTTGTGGAACAGCTG-3′ SEQ ID NO: 244  
     [0080] and  
     [0081] 5′-TGG AAC GAT AAA TGC CCA TG-3′ SEQ ID NO: 245  
     [0082] and pING3354 was amplified with these primers. The approximately 550 bp amplified DNA fragment was cut with ApaI, and the 48 bp DNA fragment was purified on an agarose gel. Approximately 1 μg of 48 bp fragment was ligated in a 30 μL reaction with 5 U T4 ligase. Three 10 μL aliquots were removed at 0.5, 3 and 15 minutes and added into 2 μL of 60 mM EDTA to stop the ligation reaction. The three samples were then mixed together, and the ligation products in the size range expected for 2 to 5 repeat units were purified on an agarose gel. Plasmid pING3354 was cut with ApaI, and the DNA was dephosphorylated with Calf intestinal alkaline phosphatase. The 48 bp DNA repeat units were ligated to the pING3354 vector and used to transform  E. coli  MC1061. The resultant clones were analyzed by restriction analysis. Clones containing 2, 3, 4 and 5 repeat units were identified, and the DNA sequence of the entire repeat insert was sequenced with Sequenase™. Each clone was digested with EcoRI and XhoI. These DNA fragments were ligated into the plasmid vector pING3217 that had been digested with EcoRI and XhoI. The resultant plasmids containing 2, 3, 4, and 5 repeat units of the peptide sequence were designated pING3359, pING3360. pING3361, and pING3362, respectively. The sequences of the fusion proteins encoded by pING3359 is shown in SEQ ID NOS: 262 and 263.  
     [0083] 8. Alternative Preparation of Vector Constructs  
     [0084] The expression vectors encoding peptide fusion proteins as described above could be alternatively made by cutting pING3737/ATCC 69009 (or any of pING3746/ATCC 69008; pING3747/ATCC 69101; pING3754/ATCC 69102; pING3758/ATCC69103; pING3759/ATCC 69104; pING3336/ATCC6933 1; pING4644/ATCC 69332; pING4629/ATCC 69333) with EcoRI and XhoI and purifying the vector fragment, then preparing a synthetic DNA segment having a sequence encoding a pel B leader (see, e.g., amino acids 1-22 of SEQ ID NOS: 246 or 248) then preparing a synthetic DNA segment encoding a gelonin-peptide or Bone D-peptide fusion protein, for example, as shown in SEQ ID NOS: 250, 252. 254, 258 260 or 262 described herein.  
     EXAMPLE 2  
     Expression of Recombinant Fusion Proteins  
     [0085] 1. Lab Scale Production Process  
     [0086] Expression of a recombinant product under control of the araB promoter was evaluated as follows. Expression vector constructs are transformed into  E. coli  E104 (deposited as ATCC 69009; ATCC 69008; ATCC 69101; ATCC 69102; ATCC69103; ATCC 69104; ATCC 69331; ATCC 69332; ATCC 69333, each containing a gelonin-encoding plasmid) and bacterial cultures were grown at 37° C. in TYE medium (15 g Tryptone, 10 g Yeast Extract, 5 g NaCl per liter) supplemented with 15 μg/mL of tetracycline to an OD 600 ≈0.4. L-arabinose from a 20% W/V solution was added to a final concentration of 0.1%. The bacterial culture was then incubated for up to 16 hours post-induction at 37° C. Secreted products were detected directly in the cell-free culture supernatant. Cells were separated by centrifugation, and culture supernatants were filtered with a 0.2 m Acrodisc filter (Gelman) and stored at 4° C. Recombinant product was detected in the culture supernatant by ELISA or analyzed on a polyacrylamide gel. Recombinant proteins that remain associated with the cellular fraction were evaluated directly by SDS-PAGE of resuspended cell pellets.  
     [0087] 2. Scale Up Fermentation Process  
     [0088] A bacterial culture containing the product expression vector was inoculated into 100 mL of GMM culture medium described below and grown at 32° C. to approximately 200 Klett Units then inoculated into a 35 L fermenter. The final volume of the fermenter was approximately 10 liters or 20 titers containing minimal salts medium with glycerol as a carbon source (Glycerol Minimal Media, GMM). For a 10 L run, the fermenter vessel was autoclaved with 7.35L GMM final volume containing:  
                                                      (NH 4 ) 2 SO 4       101 g           KH 2 PO 4      13.2 g           K 2 HPO 4     118.7 g           MgSO 4 .7H 2 O    2.3 g           H 3 PO 4  (Conc.)    24.8 mL           Antifoam    0.8 mL           Biotin    0.01 g           Yeast Extract    38.8 g           Glycerol     155 g                      
 
     [0089] to which a filter sterilized solution in approximately 200 mL was added prior to bacterial inoculation:  
                                                          CaCl 2 .2H 2 O (10% w/v)   7.75   mL           Trace D solution*   129   mL           Thiamin HCl (10% w/v)   0.8   mL           Nicotinic Acid (1% w/v)   15.5   mL           *Trace D solution (filtered):           FeCl 3 .6H 2 O   6.480   g           ZnSO 4 .7H 2 O   1.680   g           MnCl 2 .4H 2 O   1.200   g           Na 2 MoO 4 .2H 2 O   0.576   g           CuSO 4 .5H 2 O   0.240   g           CoCl 2 .6H 2 O   0.240   g           H 3 BO 3     0.720   g           H 3 PO 4  (Conc.)   96.0   mL           H 2 O (Batch Volume)   2.000   L                      
 
     [0090] The inoculated fermenter was maintained at pH 6.0 and 32° C. with 10 L/min air and agitation at 1000 rpm. When nutrients became limiting (as judged by an increase in DO to roughly 100%), the culture was fed with additional nutrients until the culture reached an optical density (OD 600 ) of about 40-100 (DO is kept at approximately 20%). Specifically, the culture was fed with the first AI (After Inoculation) feed:  
                                  1st AI feed:                                         Autoclaved ingredients:                   Glycerol   1960   g           MgSO 4 .7H 2 O   29.4   g           Biotin   0.026   g           H 2 O (Batch volume   2.800   L           Filtered ingredients:           CaCl 2 .2H 2 O (10% w/v)   98.1   mL           Thiamine HCl (10% w/v)   9.8   mL           Nicotinic Acid (1% w/v)   19.7   mL                             2nd AI feed:                                         Autoclaved ingredients:                   Glycerol   420   g           MgSO 4 .7H 2 O   6.3   g           Biotin   0.005   g           Arabinose   50   g           dl-H 2 O (Batch volume)   0.6   L           Filtered ingredients:           CaCl 2 .2H 2 O (10% w/v)   21   mL           Thiamine HCl (10% w/v)   2.1   mL           Nicotinic Acid (1% w/v)   4.2   mL                      
 
     [0091] The culture was induced by gradient induction at OD of approximately 40-100 with the second feed containing the inducing agent L-arabinose. Specifically, the second AI feed was:  
                               2nd AI feed:                                                    Autoclaved ingredients:                   Glycerol   420   g           MgSO 4 .7H 2 O   6.3   g           Biotin   0.005   g           Arabinose   50   g           dl-H 2 O (Batch volume)   0.6   L           Filtered ingredients:           CaCl 2 .2H 2 O (10% wlv)   21   mL           Thiamine HCl (10% w/v)   2.1   mL           Nicotinic Acid (1% w/v)   4.2   mL                      
 
     [0092] The culture was harvested 20 to 36 hours post induction.  
     [0093] The cells were separated from the culture supernatant with a 0.2 μm Microgon Hollow Fiber cartridge (10 ft. 2 ). The cell paste obtained was processed according to Example 3 below for the isolation of inclusion bodies from cells expressing an intracellular recombinant fusion protein product. Alternatively, for expressed and secreted products, the cell-free fermentation broth was concentrated and diafiltered with 10 mM sodium phosphate buffer pH 7.0 using a DC 10 with a S10Y10 Amicon cartridge. The concentrated culture medium containing the secreted recombinant product was in a volume of approximately 3 liters. The concentrated medium was processed according to Example 4 below for the isolation of recombinant fusion proteins and purification of recombinant peptides.  
     EXAMPLE 3  
     Isolation of Inclusion Bodies from Cells Expressing Intracellular Recombinant Product  
     [0094] Experiments were conducted to express a fusion protein comprised of Bone D and a BPI-derived peptide linked by an Asp-Pro peptide bond. Cell paste from  E. coli  cultured as described in Example 2 for the expression of a fusion protein of Bone D with a BPI-derived peptide (e.g., pING3353) was suspended in 100 mM Tris-HCl pH 8.0, 5 mM EDTA (7 mL/g cell paste) and incubated on ice for 10 minutes. Lysozyme (10 mg/mL in 100 mM Tris-HCl pH 8.0, 5 mM EDTA) was added to give a final concentration of 10 mg lysozyme/g cell paste, and the sample was incubated on ice until lysis occurred (i.e., when the solution became very viscous). Lysis typically occurred within about 10 minutes. The mixture was sonicated with 3 or 4 ten second pulses at the highest setting using a Sonic U sonicator (B. Braun Biotech Inc., Allentown, Pa.). The inclusion bodies were pelleted by centrifugation at 22,000 g for 40 minutes. A series of washes with Triton X-100, then 60 mM HCl, and then water followed. First the inclusion body pellet was washed by resuspending in 100 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1% Triton X-100, followed by centrifugation at 22,000 g for 40 minutes. The next washes were done additionally with 60 mM HCl and water. For each wash [at 4° C.], the resuspension of the inclusion bodies was done with a Polytron® (Brinkmann Instruments, Westbury, N.Y.). The acid wash was done to remove lysozyme from the inclusion body pellet. No recombinant peptide was released (i.e., no appearance in either the supernatant or pellet after washing the inclusion body pellet with HCl) indicating that the acid wash conditions were mild enough such that cleavage of the Asp-Pro bond in the fusion protein did not occur. Washing the inclusion body pellet with water instead of acid did not remove lysozyme.  
     [0095] Samples were analyzed by SDS-PAGE using 10-20% Tricine gels (Novex, San Diego, Calif.). Samples were prepared by boiling in SDS loading buffer with reducing agent for 5 minutes. Samples were also analyzed by HPLC, using a Beckman instrument with Shimadzu auto injector and a Vydac C 18 (#218TP54) column. Solvent A was 10% acetonitrile/0.05% TFA: solvent B was 90% acetonitrile/0.05% TFA. The column was run with an 18-40% B gradient over 20 minutes, at a flow rate of 1 mL/minute with peptide detection at 229 nm.  
     [0096] The fusion protein of Bone D and peptide was designed with an Asp-Pro peptide bond at the junction so the peptide might be liberated with acid treatment. Peptide bonds of aspartyl residues may be cleaved in dilute acid at rates at least 100 times greater than other peptide bonds and aspartyl-prolyl bonds are the most labile of the aspartyl peptide bonds. It was not known whether the inclusion bodies would need to be solubilized in order for this reaction to be effective on the fusion protein. Even if the acid was effective on the inclusion bodies without solubilization and the Asp-Pro bond of the fusion protein was cleaved, it was not known whether the free peptide and/or the Bone D protein would become soluble.  
     [0097] The acid hydrolysis of the inclusion body pellet was done with 30 mM or 60 mM HCl, but at a much higher temperature and for a longer period of time than the acid wash of inclusion bodies. Conditions of temperature and incubation time sufficient to cleave the Asp-Pro bond in the fusion protein to liberate free peptide were determined by experimentation as follows.  
     [0098] Several conditions were tested for achieving complete cleavage of the Asp-Pro bond. At 55° C. using 60 mM HCl, complete cleavage occurred by about 48 hours. This was evident from SDS-PAGE analysis; after 48 hours the fusion protein band had nearly disappeared as it was converted into Bone D and peptide. Samples were analyzed by SDS-PAGE and the gel results from the pellet after acid hydrolysis indicated that Bone D protein surprisingly remained insoluble after the 60 mM HCl treatment. This result was advantageous because it allowed isolation of the cleaved peptide from the acid supernatants. Specifically, SDS-PAGE analysis of the supernatants after acid hydrolysis revealed that the peptide was found soluble in the supernatant after cleavage from the insoluble fusion protein. If the inclusion body pellet was not washed with acid to remove lysozyme as described above, a 14 kD lysozyme band was observed in the inclusion body pellet by SDS-PAGE.  
     [0099] SDS-PAGE analysis revealed the time-dependent generation of peptide in the supernatant during acid hydrolysis. In an attempt to reduce the time required for complete hydrolysis of the Asp-Pro bond in the fusion protein, higher temperature was used. Peptide was liberated from the fusion protein over time at 85° C. using 60 mM HCl. The reaction was complete between 2 and 4 hours since the fusion protein was not present in the 4 hour sample analyzed by SDS-PAGE. The accumulation of peptide in the supernatant was observed over time, however, after 2 hours a slight decrease in the peptide concentration was observed presumably due to peptide hydrolysis and/or deamidation. Acid hydrolysis was also conducted at higher temperature but with a lower HCl concentration of 30 mM. Under these conditions at 85° C. the reaction was complete at 4 hours. Fusion protein was not present at 4 hours by SDS-PAGE analysis. By 6 hours, the peptide concentration began to decrease presumably due to peptide hydrolysis and/or deamidation.  
     [0100]FIG. 1 shows the acid liability of the Asp-Pro peptide linker over time at various temperatures. FIG. 2 shows an Arrhenius plot for the hydrolysis of fusion protein, demonstrating that the rate of hydrolysis is proportional to the temperature.  
     [0101] Additional experiments were performed to investigate conditions for complete acid cleavage of fusion protein from inclusion bodies with 30 mM HCl at 85° C. but using various weight/volume concentrations of inclusion bodies. For scale-up processes, it was desirable to be able to use high weight/volume concentration of inclusion bodies, however, in the experiments described above and in previously published experiments with a very different recombinant fusion protein [REF], the highest weight/volume concentrations used for cleavage were 10% (i.e., 10 g wet/weight per 100 mL volume) and 6% (i.e., 6 g per 100 mL), respectively.  
     [0102] The results shown in FIG. 3 demonstrate while maximal acid cleavage was achieved with a 10% weight/volume at 85° C. in 4-6 hours, when the weight/volume concentrations of inclusion bodies were increased to 20%, 30% and 50%, the reaction rate decreased significantly. In particular, at the 30% and 50% concentrations little or no peptide was released by acid hydrolysis of the inclusion bodies. When the pH of each of the acid-treated inclusion body suspensions was measured, it was discovered that the pH of the suspensions varied dramatically (pH 2.6, 3.7, 4.4 and 5.1 for acid-treated inclusion body suspensions of 10%, 20%, 30% and 50%, respectively.  
     [0103] In order to test whether the variation in pH was the cause of the decreased release, and if so what pH range might be critical for achieving complete hydrolysis, additional experiments were performed using 30% inclusion body suspensions using 30 mM, 60 mM or 90 mM HCl. As shown in FIG. 4, maximal peptide release was achieved with the 60 mM and 90 mM HCl treated suspensions. The pH of the 30 mM, 60 mM and 90 mM treated suspension was 3.5, 2.4 and 1.5, respectively. The conclusion from these experiments was that obtaining and maintaining a low pH suspension of inclusion bodies was critical to achieving maximal peptide cleavage and release. Presently preferred conditions for acid hydrolysis include titration to pH 2.2 with concentrated HCl using suspensions that are greater than 10% in weight/volume concentration, preferably 30%. Having discovered that achieving a constant pH of &lt;2.6 is important for efficient and complete acid hydrolysis for the release of peptide, high weight/volume concentrations (i.e., &gt;10-50%) of inclusion bodies may be routinely and efficiently processed.  
     [0104] For the purification of recombinant peptides from  E. coli , three column separation steps were utilized to achieve sufficiently low levels of impurities (e.g., protein, endotoxin, and DNA). After acid hydrolysis, the supematant was neutralized. However, in initial experiments, a precipitate formed which included some of the peptide. This precipitation could be avoided by addition of &gt;5 M urea.  
     [0105] For initial attempts to work out a process, 30 g of cell paste were utilized. SP sepharose was chosen for the first step. As a second column step, the hydrophobic interaction resin butyl sepharose was chosen. Gel filtration chromatography with Superdex 30 (Pharmacia) was chosen as the last step. As shown in Table 1, the overall recovery was 10.7%, with most of the loss occurring at the SP Sepharose step. Mass spectrum analysis of the peptide isolated from this purification showed a mass (3735) consistent with the predicted mass (3735.7) indicating an intact amino and carboxyl terminus. The antimicrobial activities of the purified peptide were assayed as described in Example 5, and found to be active.  
     [0106] Based on results from additional experiments, a purification process with 30 g of cell paste was tested which incorporated an ultrafiltration step in 5M urea followed by a CM Spherodex column. As shown in Table 2, the recovery for this purification scheme was 31%. Most of the loss occurred at the filtration step.  
                           TABLE 1                                   %                   Recovery               %   From           Volume   Recovery   Previous       Sample   (mL)   by HPLC   Step                                                Supernatant from HCl treatment   202   100.0           SP Sepharose flow-through   284   0.0       SP Sepharose eluate   111   13.2   13.2       SP Sepharose strip   184   37.0       Butyl Sepharose flow-through   377   0.0       Butyl Sepharose eluate   31   11.9   89.9       Butyl Sepharose strip   0   0.0       Superdex 30 pool (final)   20   10.7   89.8                  
 
     [0107]                           TABLE 2                                   %                   Recovery               %   From           Volume   Recovery   Previous       Sample   (mL)   by HPLC   Step                                                Supernatant from HCl treatment   200   100.0           100K flow through   400   41.8   41.8       CM Spherodex flow through   440   0.0       CM Spherodex eluate   67   33.1   79.2       CM Spherodex strip   33   0.0       Butyl Sepharose eluate   37   29.6   89.5       Superdex 30 pool (final)   19.5   30.9   104.4                    
     [0108] Additional experiments were conducted to express a fusion protein comprising Bone D and multiple repeats of a BPI-derived peptide linked by Asp-Pro peptide bonds. For these experiments, five fermentation batches in a 35 liter vessel were grown, each producing a different fusion protein. Specifically, five fermentation batches were grown in a 35 L fermenter as described in Example 2 of  E. coli  E104 cells containing plasmids pING3796 (single peptide construct), pING3359 (2 peptide construct), pING3361 (3 peptide construct), pING3360 (4 peptide construct) and pING3362 (5 peptide construct), thus, increasing numbers of peptide units were produced in each batch. In an experiment, one gram of cell paste from each fermentation batch was lysed by lysozyme/EDTA followed by sonication as described above. The inclusion bodies were isolated by centrifugation and suspended in 30 mM HCl for 3.5 hours at 85° C. Peptide in the supernatant was quantitated by HPLC. Duplicate aliquots from each fermentation were analyzed. Results as shown in FIG. 5 revealed that the product from pING3360 with four peptide repeat units had the highest yield.  
     [0109] Mechanical description of the cells in the fermentation batches described above was investigated as an alternative to the procedure described in this Example 3, using lysozyme/EDTA followed by sonication. Mechanical methods for cell disruption are well suited for large scale applications, thus the use of a Microfluidizer® (Microfluidics International Corporation, Newton, Mass., model M-11OY) for large scale cell lysis was investigated. With this device, product is driven by air pressure through microchannels at high velocity. The process stream is split and then reunited allowing the cells to collide. The resulting shear and cavitation forces disrupt the cells. Greater than 95% lysis was achieved with a single pass. Process times were about 30 minutes for 10 liters of resuspended cell paste with a density of 10% suspended solids. Inclusion bodies were isolated by centrifugation at 11,000 g for 1 hour.  
     [0110] Complete hydrolysis of aspartyl prolyl bonds was achieved by suspending inclusion bodies obtained from each of the 5 fermentation batches in 30 mM HCl [10%, weight/volume] and incubating at 85° C. for 4-5 hours as described above. Alternatively, inclusion bodies could be resuspended in water followed by acid addition. Released peptide was soluble in the aqueous environment. The acid hydrolysate was neutralized by adding sodium citrate to buffer the solution and then NaOH to adjust the pH to 6.0. Using this mechanical description method, no precipitation was observed with neutralization after acid hydrolysis as was seen with the lysozyme/EDTA/sonication method. Numerous  E. coli  protein impurities in the acid hydrolysate were removed by subsequent purification steps.  
     [0111] Experiments were done to determine the optimal elution conditions for the two columns used in the purification process. For the CM Sepharose column, sample was loaded and the column was step eluted with increasing concentrations of NaCl in 10 mM citrate buffer at pH 3.0. Most of the peptide eluted at 40 mM NaCl with a small amount eluting at 80 mM NaCl. Therefore, the elution buffer for CM Sepharose was chosen as 10 mM citrate, 80 mM NaCl, pH 3.0. Elution conditions for the butyl sepharose column were determined by similar experiments. A butyl sepharose column loaded with peptide was step eluted with decreasing concentrations of ammonium sulfate in 10 mM sodium phosphate buffer at pH 7.0. Relatively pure peptide eluted at ammonium sulfate concentrations down to 1.1 M. Impurities began to elute with the peptide at 0.8 M ammonium sulfate. In these experiments, the optimal ammonium sulfate concentration for achieving pure peptide was between 0.8 M and 1.1 M.  
     [0112] When peptide was purified from 5 grams of inclusion bodies using these optimized elution conditions, SDS-PAGE analysis revealed that the pellet after acid hydrolysis contained predominantly Bone D and very little fusion protein indicating that the hydrolysis was nearly complete. Numerous protein impurities were present in the acid hydrolysate. A significant purification was achieved on the CM sepharose column and after butyl sepharose there were no protein impurity bands detected by SDS-PAGE.  
     [0113] Purity was also assessed by HPLC analysis using a Beckman instrument with Shimadzu auto injector and a C18 (Vydac, #218TP54) reverse phase column. Solvent A was  10  % acetonitrile/0.1% TFA; solvent B was 90% acetonitrile/0.1% TFA. The C18 column was run with a 15%-35% B gradient over 20 minutes, with a 1 mL/minute flow rate. Peptide detection at 229 nm. The deamidated form of the peptide was present in the acid supernatant at levels of approximately 5% and was not removed by the purification process.  
     [0114] As shown in Table 3, recovery after the CM sepharose and butyl sepharose columns was 87.2% and 43.5%, respectively. Recombinant peptides isolated from  E. coli  by this method tested as described in Example 5 and found to be as active in a radial diffusion assay as the comparable synthetic peptides.  
     [0115] In an effort to further improve the purification process, a resin which could substitute for butyl sepharose was selected for additional experiments. A CM sepharose eluate was loaded onto a Source reverse phase resin (Pharmacia) column and then eluted with a gradient of increasing acetonitrile concentration. The peptide peak was isolated and the solvent removed by evaporation in a vacuum centrifuge. Sample was resuspended in buffer and evaluated for purity by SDS-PAGE and HPLC. Purity was comparable to that obtained with a butyl sepharose column with improved yield. The yield at this column step was 98%, which was a significant improvement over the butyl sepharose column.  
                               TABLE 3                                       Volume               Sample   (mL)   % Recovery                                                        HCl supernatant   45   100.0           HCl supernatant neutralized   45   92.9           CM sepharose flow-through   99   0.0           CM sepharose eluate   47   87.2           CM sepharose strip   36   0.9           Butyl Sepharose flow-through   140    3.7           Butyl Sepharose eluate   45   43.5           Butyl sepharose strip   26   2.7                      
 
     EXAMPLE 4  
     Isolation of Secreted Fusion Protein and Purification of Recombinant Peptide from Bacterial Host Cell Culture Media  
     [0116] E. coli  E 104 cells containing pING3797 encoding a peptide-gelonin fusion protein was grown in a fermenter as described in Example 2. The secreted fusion protein was isolated from the  E. coli  fermentation broth after cell growth. The fermentation broth was separated from the bacterial cells and the cell-free fermentation broth was concentrated and diafiltered with 10 MM sodium phosphate pH 7.0 using a DC10 with an S10Y10 Amicon cartridge as described in Example 2. The concentrated culture medium was in a volume of approximately 3 liters. Diafiltered material was loaded onto a column of CM Sepharose Fast Flow (Pharmacia, Uppsala, Sweden) equilibrated in 10 mM sodium phosphate, pH 7.0. The column was eluted in 10 mM sodium phosphate, 400 mM NaCl, pH 7.0 to isolate the fusion protein. The gelonin-peptide fusion protein was approximately 70% pure and was found to be active in a radial diffusion assay as described in Example 5.  
     [0117] Concentrated HCl was added to the CM Sepharose eluate to a final HCl concentration of 30 mM. The sample was incubated at 85° C. for 3 hours and then neutralized by adding 500 mM sodium citrate to a final concentration of 15 mM. A precipitate which formed was removed by centrifugation and the sample S was further purified by HPLC on a C18 column. The peptide was eluted with a 15-35% gradient of 90% acetonitrile/0.05% TFA over 20 minutes at a flow rate of 1 mL/minute. The peptide peak was isolated and its identity was confirmed by N-terminal sequence analysis. The antimicrobial activity of the purified peptide was determined by radial diffusion assay as described in Example 5 below.  
     EXAMPLE 5  
     Radial Diffusion Assays for Antimicrobial Activity Analysis of Recombinant Peptides  
     [0118] A variety of BPI-derived peptides, including those comprising the sequences listed in Table 4 (SEQ ID NOS: 1-239) may be produced by recombinant methods of the invention and tested for antimicrobial activity (both anti-fungal and anti-bacterial activity) in radial diffusion assays. Experiments were initially performed to assess the antifungal activity of the recombinantly produced peptides in a radial diffusion assay. For these experiments,  Saccharomyces cerevisiae  PS6 or  Candida albicans  SLU-1 were added at 1×10 6  cells per mL into 8 mL of {fraction (1/100)}× Sabouraud dextrose broth in 1% agarose plus 300 mM EDTA and 0.02% Tween 20. The mixture was poured into a plate and 3.5 mM wells were made by hole punches after solidification. Samples were diluted into saline, and 5 μL samples were added to each well. The samples were incubated at room temperature for three hours and then the plates were overlayed with 8 mL of 2× Sabouraud dextrose broth in 1% agarose plus 300 mM EDTA and 0.02% Tween 20. Areas of inhibition were calculated after a 24 hour incubation at 30° C. Peptide prepared as described in Example 3 was found to be active in this assay. Fusion protein prepared as described in Example 4 was also found to be active in this assay.  
     [0119] To assess the antibacterial activity of the recombinantly produced peptides, additional experiments are conducted with  E. coli  J5 or  E. coli  E104 cells in a radial diffusion assay. For these experiments, cultures of  E. coli  J5 were grown overnight in TYE broth (15 g tryptone, 10 g yeast extract, 5 g NaCl per liter) and then grown to mid logarithmic phase in TEA broth media (Simon et al.,  Proc. Nat&#39;l. Acad. Sci.  ( USA ), 51:877-883 (1964)).  E. coli  J5 cells at 2-3×10 5  cells/mL were added to molten 0.8% agarose containing nutrient broth. Serial dilutions of peptides were prepared in 0.15 M NaCl. Five μl of the diluted peptides, or as a control, saline alone, were added to 3 mm wells prepared in the hardened agarose (5 μl/well). The plates were sealed with parafilm to prevent drying and incubated at 37° C. for 24 hours. Zones of inhibition were measured and the net areas of inhibition determined by subtracting the area comprising the 3 mm well from the area encompassing the 3 mm well plus the inhibition zone. Recombinantly produced peptides are assayed for antibacterial activity and compared to the equivalent synthetic peptides. When the synthetic peptides XMP.13, XMP.284, XMP.353, XMP.366, XMP.406 and XMP.407 with sequences shown in Table 4 below were assayed for antibacterial activity peptide XMP.284 exhibited the most antibacterial activity followed by XMP. 13, XMP.391, XMP.366, XMP.353, XMP.406 and XMP.407 as the least bactericidal. Synthetic peptides XMP.406 and XMP.407 have the same sequences as peptides prepared from the encoded fusion protein described in Example 1, Section 7 and Example 3. Recombinant peptide prepared from pING3353 (Examples 1 and 3) was found to be active in this assay.  
                           TABLE 4                                   Peptide   Peptide Amino           (SEQ ID NO:)   Acid Segment                          XMP.1 (1)   19-33           XMP.2 (2)   85-99           XMP.3 (3)   73-99           XMP.4 (4)   25-46           XMP.5 (5)   142-163           XMP.6 (6)   112-127           XMP.7 (7)   (90-99) × 2           XMP.8 (8)   90-99           XMP.9 (9)   95-99, 90-99           XMP.10 (10 &amp; 11)   94-99, 90-99, 90-99 and               95-99, 90-99, 90-99           XMP.10a (10)   94-99, 90-99, 90-99           XMP.10b (11)   95-99, 90-99, 90-99           XMP.11 (12)   148-151, 153-161           XMP.12 (13)   141-169           XMP.13 (14)   148-161           XMP.14 (15)   21-50           XMP.15 (16)   85-99, A @ 85 (I)           XMP.16 (17)   85-99, A @ 86 (K)           XMP.17 (18)   85-99, A @ 87 (L)           XMP.18 (19)   85-99, A @ 88 (S)           XMP.19 (20)   85-99, A @ 89 (G)           XMP.20 (21)   85-99, A @ 90 (K)           XMP.21 (22)   85-99, A @ 91 (W)           XMP.22 (23)   85-99, A @ 92 (K)           XMP.23 (24)   85-99, A @ 94 (Q)           XMP.24 (25)   85-99, A @ 95 (K)           XMP.25 (26)   85-99, A @ 96 (R)           XMP.26 (27)   85-99, A @ 97 (F)           XMP.27 (28)   85-99, A @ 98 (L)           XMP.28 (29)   85-99, A @ 99 (K)           XMP.29 (30)   (148-161) × 2           XMP.30 (31)   90-99, 148-161           XMP.31 (32)   148-161, A @ 148 (K)           XMP.32 (33)   148-161, A @ 149 (S)           XMP.33 (34)   148-161, A @ 150 (K)           XMP.34 (35)   148-161, A @ 151 (V)           XMP.35 (36)   148-161, A @ 152 (G)           XMP.36 (37)   148-161, A @ 153 (W)           XMP.37 (38)   148-161, A @ 154 (L)           XMP.38 (39)   148-161, A @ 155 (I)           XMP.39 (40)   148-161, A @ 156 (Q)           XMP.40 (41)   148-161, A @ 157 (L)           XMP.41 (42)   148-161, A @ 158 (F)           XMP.42 (43)   148-161, A @ 159 (H)           XMP.43 (44)   148-161, A @ 160 (K)           XMP.44 (45)   148-161, A @ 161 (K)           XMP.45 (46)   148-161, A @ 161 (K)           XMP.46 (47)   (90-99) × 2, A @ 1st               94 (Q) &amp; 95 (K)           XMP.47 (48)   (90-99) × 2, A @ 2d               94 (Q) &amp; 95 (K)           XMP.48 (49)   (90-99) × 2, A @ both               94 (Q) &amp; 95 (K)           XMP.49 (50)   178-191           XMP.50 (51)   178-192           XMP.51 (52)   178-193           XMP.52 (53)   178-194           XMP.53 (54)   178-195           XMP.54 (55)   21-35           XMP.55 (56)   152-172           XMP.56 (57)   85-99, K @ 94 (Q) &amp; Q               @ 95 (K)           XMP.57 (58)   Cys 85-99 Cys           XMP.58 (59)   Cys 85-99           XMP.59 (60)   85-99, A @               90 (K) &amp; 92 (K)           XMP.60 (61)   85-99, A @               86 (K) &amp; 99 (K)           XMP.61 (62)   85-99, F @ 91 (W)           XMP.62 (63)   119-148* {circumflex over ( )}           XMP.63 (64)   85-99, 148-161           XMP.64 (65)    178-193*           XMP.65 Rd (66)   Cys-85-99-Cys           XMP.65 Ox (67)   Cys-85-99-Cys           XMP.68 (68)   119-148, ALA @               132 (C)* {circumflex over ( )}           XMP.69 (69)   [90-99, A @ 94 (Q) &amp;               95 (K)] × 3           XMP.73 (70)   85-99, F @ 95 (K)           XMP.74 (71)   148-161, 90-99           XMP.75 (72)   IKKRAISFLGKKWQK               (2-mixed)           XMP.77 (73)   85-99, W @ 95 (K)           XMP.78 (74)    100-118*           XMP.79 (75)   85-99, K @ 94 (Q)           XMP.81 (76)   85-99, F @ 94 (Q)           XMP.82 (77)   148-161, W @ 158 (F)           XMP.85 (78)   148-161, L @ 152 (G)           XMP.86 (79)   148-161, L @ 156 (Q)           XMP.87 (80)   148-161, L @ 159 (H)           XMP.88 (81)   85-99, F @ 94 (Q)           XMP.91 (82)   148-161, F @ 156               (Q)           XMP.92 (83)   148-161, K @ 156 (Q)           XMP.94 (84)   148-161, F @ 159 (H)           XMP.95 (85)   148-161, F @ 152 (G)           XMP.96 (86)   148-161, F @ 161 (K)           XMP.97 (87)   148-161, K @ 152 (G)           XMP.99 (88)   [90-99, W @ 95               (K)] × 3           XMP.100 (89)   148-161, K @ 152 (G)               &amp;               156 (Q)           XMP.101 (90)   (148-161) × 2[K @               152 (G) &amp; 156 (Q),               F @ 159 (H) &amp; 161 (K)]           XMP.102 (91)   90-99 (F @ 95 (K)) +               148-161 L @ 156 (Q)           XMP.103 (92)   85-99, W @ 94 (Q)           XMP.104 (93)   148-161, S @ 156 (Q)           XMP.106 (94)   148-161, T @ 156 (Q)           XMP.107 (95)   148-161, W @ 159 (H)           XMP.108 (96)   148-161, W @ 161 (K)           XMP.113 (97)   148-161, F @ 157 (L)           XMP.114 (98)   KWQLRSKGKIKIFKA           XMP.115 (99)   170-177* {circumflex over ( )}           XMP.117 (100)   49-59*             XMP.118 (101)   60-77*             XMP.120 (102)   85-99, K @ 97 (F)           XMP.124 (103)   148-161, K @ 152 (G),               158 (F)           XMP.125 (104)   148-161, Y @ 156 (Q)           XMP.127 (105)   148-161, F @ 153 (W)           XMP.135 (106)   148-161, K @ 153 (W)           XMP.136 (107)   85 -99, E @ 95 (K)           XMP.137 (108)   C-148-161-C           XMP.138 (109)   148-161, K @ 152 (G),               F @ 153 (W)           XMP.139 (110)   148-161, Y @ 153 (W)           XMP.141 (111)   85-99, W @ 97 (F)           XMP.142 (112)   148-161, W @ 157 (L)           XMP.147 (113)   85-99 K @ 96 (R)           XMP.149 (114)   KWKVFKKIEK + 148-               161           XMP.150 (115)   KWAFAKKQKKRLKR               QWLKKF           XMP.151 (116)   94-99, 90-99, 90-99           XMP.152 (117)   95-99, 90-99, 90-99           XMP.153 (118)   (90-99) × 3           XMP.161 (119)   148-161, K @ 152 (G)               153 (W)           XMP.162 (120)   90-99, 148-161, W @               95 (K)           XMP.163 (121)   (90-99) × 2, W @ both               95 (K)           XMP.166 (122)   148-161, V @ 153 (W)           XMP.167 (123)   90-97           XMP.168 (124)    C-90-101-C           XMP.169 (125)   C-90-97-C           XMP.170 (126)    90-101           XMP.241 (127)   148-161, L @ 156 (Q),               W @ 158 (F)           XMP.245 (128)   90-99, 148-161, F @ 95               (K), W @ 158 (F)           XMP.249 (129)   148-161, G @ 153 (W)           XMP.250 (130)   148-161, L @ 153 (W)           XMP.251 (131)   148-161, I @ 153 (W)           XMP.262 (132)   148-161, N @ 156 (Q)           XMP.263 (133)   148-161, E @ 156 (Q)           XMP.264 (134)   148-161, D @ 156 (Q)           XMP.265 (135)   148-161, R @ 156 (Q)           XMP.266 (136)   148-161, K @ 152 (G),               V @ 153 (W)           XMP.267 (137)   148-161, K @ 152 (G),               154 (L)           XMP.268 (138)   148-161, V @ 153 (W),               A @ 154 (L)           XMP.269 (139)   148-161, K @ 152 (G),               V @ 153 (W), A @ 154               (L)           XMP.270 (140)   (148-161) + (148-161), L               @ 1st 156 (Q)           XMP.271 (141)   (148-161) + (148-161), L               @ 2nd 156 (Q)           XMP.272 (142)   (148-161) + (148-161), L               @ both 156 (Q)           XMP.273 (143)   (148-161) + (148-161), F               @ 1st 156 (Q)           XMP.274 (144)   (148-161) + (148-161), F               @ 2nd 156 (Q)           XMP.275 (145)   (148-161) + (148-161), F               @ both 156 (Q)           XMP.276 (146)   90-99, 148-161, F @ 95               (K) &amp; 156 (Q)           XMP.278 (147)   85-99, A @ 94 (Q), W               @ 95 (K)           XMP.280 (148)   85-99, A @ 94 (Q), F @               95 (K)           XMP.282 (149)   [90-99, F @ 94 (Q) &amp;               95 (K)] × 2           XMP.283 (150)   148-161, K @ 152 (G),               F @ 153 (W), K @ 156               (Q)           XMP.284 (151)   149-161, K @ 152 (G)           XMP.285 (152)   149-160, K @ 152 (G)           XMP.286 (153)   150-161, K @ 152 (G)           XMP.287 (154)   149-159, K @ 152 (G)           XMP.288 (155)   150-160, K @ 152 (G)           XMP.289 (156)   151-161, K @ 152 (G)           XMP.290 (157)   149-158, K @ 152 (G)           XMP.291 (158)   150-159, K @ 152 (G)           XMP.292 (159)   151-160, K @ 152 (G)           XMP.293 (160)   152-161, K @ 152 (G)           XMP.294 (161)   149-157, K @ 152 (G)           XMP.295 (162)   150-158, K @ 152 (G)           XMP.296 (163)   151-159, K @ 152 (G)           XMP.297 (164)   152-160 K @ 152 (G)           XMP.298 (165)   153-161           XMP.299 (166)   149-156, K @ 152 (G)           XMP.300 (167)   150-157, K @ 152 (G)           XMP.301 (168)   151-158, K @ 152 (G)           XMP.302 (169)   152-159, K @ 152 (G)           XMP.303 (170)   153-160           XMP.304 (171)   154-161           XMP.305 (172)   149-155, K @ 152 (G)           XMP.306 (173)   150-156, K @ 152 (G)           XMP.307 (174)   151-157, K @ 152 (G)           XMP.308 (175)   152-158, K @ 152 (G)           XMP.309 (176)   153-159           XMP.310 (177)   154-160           XMP.311 (178)   155-161           XMP.312 (179)   149-154, K @ 152 (G)           XMP.313 (180)   150-155, K @ 152 (G)           XMP.314 (181)   151-156, K @ 152 (G)           XMP.315 (182)   152-157, K @ 152 (G)           XMP.316 (183)   153-158           XMP.317 (184)   154-159           XMP.318 (185)   155-160           XMP.319 (186)   156-161           XMP.320 (187)   153-157           XMP.321 (188)   153-157 - K           XMP.322 (189)   153-157 - K - K           XMP.323 (190)   K - 153-157 - K           XMP.324 (191)   K - 153-157 - K - K           XMP.325 (192)   K - K - 153-157           XMP.326 (193)   K - K - 153-157 - K           XMP.327 (194)   K - K - 153-157 - K -               K           XMP.328 (195)   Pro - (85-99) +               (148-162)*           XMP.330 (196)   153-156           XMP.331 (197)   †K - K - 153-157 -K -               K           XMP.335 (198)   P - K - 153-157 - K -               K           XMP.336 (199)   R - R - 153-157 - R - R           XMP.337 (200)   H - H - 153-157 - H -               H           XMP.343 (201)   K - K - 153-157 - K -               K, V @ 153 (W)           XMP.344 (202)   K - K - 153-157 - K -               K, A @ 154 (L)           XMP.345 (203)   K - K - 153-157 - K -               K, A @ 157 (L)           XMP.348 (204)   K - K - K - 153-157 -               K - K           XMP.349(205)   K - K - 153-157 - K -               K - K           XMP.350 (206)   K - K - K - 153-157 -               K - K - K           XMP.351 (207)   K - K - 153-158 - K -               K           XMP.352 (208)   K - K - 153-161           XMP.353 (209)   P - 153-161*           XMP.354 (210)   †P - 153-161*           XMP.355 (211)   P - 153-161           XMP.356 (212)   †P - 153-161           XMP.357 (213)   K - 153-160 - P           XMP.358 (214)   K - K - 153-160 - P           XMP.373 (215)   †152-161, K @ 152 (G)           XMP.377 (216)   K - K - K - W - A - I -               Q - L - K - K           XMP.378 (217)   P - W - A - I - Q - L -               K - K           XMP.379 (218)   K - K - P -W - A - I -               Q - L - K - K           XMP.380 (219)   K - K - Q - L - L - L -               L - K - K           XMP.381 (220)   K - K - L - Q - L - L -               L - K - K           XMP.382 (221)   K - K - L - L - Q - L -               L - K - K           XMP.383 (222)   K - K - L - L - L - Q -               L - K - K           XMP.384 (223)   K - K - L - L - L - L -               Q - K - K           XMP.385 (224)   K - K - L - L - L - L -               L - K - K           XMP.386 (225)   152-161; K @ 152 (G),               A @ 154 (L)           XMP.387 (226)   152-161, P @ 152 (G),               A @ 154 (L)           XMP.388 (227)   152-161           XMP.389 (228)   151-161, K @ 151 (V)           XMP.390 (229)   151-161, K @ 151 (V),               P @ 152 (G)           XMP.391 (230)   150-161           XMP.392 (231)   150-161, P @ 152 (G)           XMP.393 (232)   148-161, P @ 152 (G)           XMP.399 (233)   148-161, F @ 156 (Q),               W @ 158 (F)           XMP.406 (234)   147-161, P @ 147 (S),               A @ 153(W)           XMP.407 (235)   147-162, P @ 147 (S),               A @ 153 (W), D @ 162               (I)           XMP.409 (236)   S - K - 153-157 - K -               K, A @ 154 (L)           XMP.412 (237)   L - K - K - K - W - A - I -               Q           XMP.413 (238)   90-96, 98-99, 90-99, A @               both 94 (Q) &amp; 95 (K)           XMP.418 (239)   148-150, 152-161, P @               152 (G)                      
 
     EXAMPLE 6  
     Additional Biological Activity Assays of Recombinant Peptides  
     [0120] To assess the endotoxin binding and neutralizing activity of the recombinantly produced peptides, additional experiments are conducted using assays, including a RAW cell-based assay as described in co-owned and copending U.S. patent application Ser. No. 08/372,105 and WO95/19179 (PCT/US95/00498), incorporated by reference in its entirety (see, e.g., Example 7).  
     [0121] To assess the heparin binding and neutralizing activity of the recombinantly produced peptides, additional experiments are performed using assays, including a TCT clotting in co-owned assay, as described U.S. Pat. No. 5,348,942 and in co-owned and copending U.S. patent application Ser. No. 08/306,473 and WO95/19372 (PCT/US94/10427), incorporated by reference in their entirety.  
     [0122] It should be understood that the foregoing disclosure emphasizes certain specific embodiments of the invention and all modifications or alternatives equivalent thereto are within the spirit and scope of the invention as set forth in the appended claims. In particular, numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing description on the presently preferred embodiments thereof. Consequently the only limitations which should be placed upon the scope of the present invention are those that appear in the appended claims.   
    
     
       
         1 
         
           
             
265 
 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.1”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              1 

Gln Gln Gly Thr Ala Ala Leu Gln Lys Glu Leu Lys Arg Ile Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.2”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              2 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               27 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.3”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              3 

Asn Val Gly Leu Lys Phe Ser Ile Ser Asn Ala Asn Ile Lys Ile Ser 
1               5                   10                  15 

Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
            20                  25 

 
           
           
             
               22 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.4”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              4 

Leu Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro Asp Tyr Ser Asp Ser 
1               5                   10                  15 

Phe Lys Ile Lys His Leu 
            20 

 
           
           
             
               22 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.5”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              5 

Val His Val His Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gln Leu 
1               5                   10                  15 

Phe His Lys Lys Ile Glu 
            20 

 
           
           
             
               17 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.6”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              6 

Ser Ile Ser Ala Asp Leu Lys Leu Gly Ser Asn Pro Thr Ser Gly Lys 
1               5                   10                  15 

Pro 

 
           
           
             
               20 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.7”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              7 

Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Lys Trp Lys Ala Gln Lys 
1               5                   10                  15 

Arg Phe Leu Lys 
            20 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.8”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              8 

Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.9”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              9 

Lys Arg Phe Leu Lys Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               25 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.10a”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              10 

Lys Arg Phe Leu Lys Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Lys 
1               5                   10                  15 

Trp Lys Ala Gln Lys Arg Phe Leu Lys 
            20                  25 

 
           
           
             
               26 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.10b”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              11 

Gln Lys Arg Phe Leu Lys Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
            20                  25 

 
           
           
             
               13 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.11”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              12 

Lys Ser Lys Val Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               29 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.12”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              13 

Ser Val His Val His Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gln 
1               5                   10                  15 

Leu Phe His Lys Lys Ile Glu Ser Ala Leu Arg Asn Lys 
            20                   25 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.13”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              14 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               30 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.14”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              15 

Gly Thr Ala Ala Leu Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro Asp 
1               5                   10                  15 

Tyr Ser Asp Ser Phe Lys Ile Lys His Leu Gly Lys Gly His 
            20                  25                  30 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.15”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              16 

Ala Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.16”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              17 

Ile Ala Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.17”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              18 

Ile Lys Ala Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.18”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              19 

Ile Lys Ile Ala Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.19”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              20 

Ile Lys Ile Ser Ala Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.20”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              21 

Ile Lys Ile Ser Gly Ala Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.21”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              22 

Ile Lys Ile Ser Gly Lys Ala Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.22”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              23 

Ile Lys Ile Ser Gly Lys Trp Ala Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.23”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              24 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Ala Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.24”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              25 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Ala Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.25”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              26 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Ala Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.26”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              27 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Ala Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.27”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              28 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Ala Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.28”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              29 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Ala 
1               5                   10                  15 

 
           
           
             
               28 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.29”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              30 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys Lys Ser 
1               5                   10                  15 

Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
            20                  25 

 
           
           
             
               24 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.30”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              31 

Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Lys Ser Lys Val Gly Trp 
1               5                   10                  15 

Leu Ile Gln Leu Phe His Lys Lys 
            20 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.31”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              32 

Ala Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.32”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              33 

Lys Ala Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.33”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              34 

Lys Ser Ala Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.34”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              35 

Lys Ser Lys Ala Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.35”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              36 

Lys Ser Lys Val Ala Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.36”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              37 

Lys Ser Lys Val Gly Ala Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.37”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              38 

Lys Ser Lys Val Gly Trp Ala Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.38”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              39 

Lys Ser Lys Val Gly Trp Leu Ala Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.39”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              40 

Lys Ser Lys Val Gly Trp Leu Ile Ala Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.40”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              41 

Lys Ser Lys Val Gly Trp Leu Ile Gln Ala Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.41”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              42 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Ala His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.42”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              43 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe Ala Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.43”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              44 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Ala Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.44”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              45 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Ala 
1               5                   10 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.45”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              46 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Ala Ala Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               20 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.46”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              47 

Lys Trp Lys Ala Ala Ala Arg Phe Leu Lys Lys Trp Lys Ala Gln Lys 
1               5                   10                  15 

Arg Phe Leu Lys 
            20 

 
           
           
             
               20 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.47”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              48 

Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Lys Trp Lys Ala Ala Ala 
1               5                   10                  15 

Arg Phe Leu Lys 
            20 

 
           
           
             
               20 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.48”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              49 

Lys Trp Lys Ala Ala Ala Arg Phe Leu Lys Lys Trp Lys Ala Ala Ala 
1               5                   10                  15 

Arg Phe Leu Lys 
            20 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.49”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              50 

Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln 
1               5                   10 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.50”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              51 

Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln Thr 
1               5                   10                  15 

 
           
           
             
               16 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.51”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              52 

Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln Thr Leu 
1               5                   10                  15 

 
           
           
             
               17 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.52”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              53 

Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln Thr Leu 
1               5                   10                  15 

Pro 

 
           
           
             
               18 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.53”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              54 

Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln Thr Leu 
1               5                   10                  15 

Pro Val 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.54”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              55 

Gly Thr Ala Ala Leu Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro 
1               5                   10                  15 

 
           
           
             
               21 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.55”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              56 

Gly Trp Leu Ile Gln Leu Phe His Lys Lys Ile Glu Ser Ala Leu Arg 
1               5                   10                  15 

Asn Lys Met Asn Ser 
            20 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.56”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              57 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Lys Gln Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               16 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.57 reduced”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              58 

Cys Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Pro Leu Cys 
1               5                   10                  15 

 
           
           
             
               16 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.58 reduced”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              59 

Cys Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.59”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              60 

Ile Lys Ile Ser Gly Ala Trp Ala Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.60”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              61 

Ile Ala Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Ala 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.61”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              62 

Ile Lys Ile Ser Gly Lys Phe Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               30 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.62 reduced”
 
             
              63 

Leu Gly Ser Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cys Ser Ser 
1               5                   10                  15 

Cys Ser Ser His Ile Asn Ser Val His Val His Ile Ser Lys 
            20                  25                  30 

 
           
           
             
               29 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.63”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              64 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Lys 
1               5                   10                  15 

Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
            20                  25 

 
           
           
             
               16 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.64”
 
             
              65 

Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln Thr Leu 
1               5                   10                  15 

 
           
           
             
               17 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.65 reduced”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
             
               Disulfide-bond  
                1..17 

 
             
              66 

Cys Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

Cys 

 
           
           
             
               30 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.68 reduced”
 
             
              67 

Leu Gly Ser Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Ala Ser Ser 
1               5                   10                  15 

Cys Ser Ser His Ile Asn Ser Val His Val His Ile Ser Lys 
            20                  25                  30 

 
           
           
             
               30 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.69”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              68 

Lys Trp Lys Ala Ala Ala Arg Phe Leu Lys Lys Trp Lys Ala Ala Ala 
1               5                   10                  15 

Arg Phe Leu Lys Lys Trp Lys Ala Ala Ala Arg Phe Leu Lys 
            20                  25                  30 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.73”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              69 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Phe Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               24 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.74”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              70 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys Lys Trp 
1               5                   10                  15 

Lys Ala Gln Lys Arg Phe Leu Lys 
            20 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.75”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              71 

Ile Lys Lys Arg Ala Ile Ser Phe Leu Gly Lys Lys Trp Gln Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.77”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              72 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Trp Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               19 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.78”
 
             
              73 

Met Ser Gly Asn Phe Asp Leu Ser Ile Glu Gly Met Ser Ile Ser Ala 
1               5                   10                  15 

Asp Leu Lys 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.79”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              74 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Lys Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.81”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              75 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Phe Lys Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.82”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              76 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Trp His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.85”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              77 

Lys Ser Lys Val Leu Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.86”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              78 

Lys Ser Lys Val Gly Trp Leu Ile Leu Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.87”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              79 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe Leu Lys Lys 
1               5                   10 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.88”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              80 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Phe Phe Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.91”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              81 

Lys Ser Lys Val Gly Trp Leu Ile Phe Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.92”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              82 

Lys Ser Lys Val Gly Trp Leu Ile Lys Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.94”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              83 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe Phe Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.95”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              84 

Lys Ser Lys Val Phe Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.96”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              85 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Phe 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.97”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              86 

Lys Ser Lys Val Lys Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               30 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.99”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              87 

Lys Trp Lys Ala Gln Trp Arg Phe Leu Lys Lys Trp Lys Ala Gln Trp 
1               5                   10                  15 

Arg Phe Leu Lys Lys Trp Lys Ala Gln Trp Arg Phe Leu Lys 
            20                  25                  30 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.100”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              88 

Lys Ser Lys Val Lys Trp Leu Ile Lys Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               28 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.101”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              89 

Lys Ser Lys Val Lys Trp Leu Ile Lys Leu Phe Phe Lys Phe Lys Ser 
1               5                   10                  15 

Lys Val Lys Trp Leu Ile Lys Leu Phe Phe Lys Phe 
            20                  25 

 
           
           
             
               24 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.102”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              90 

Lys Trp Lys Ala Gln Phe Arg Phe Leu Lys Lys Ser Lys Val Gly Trp 
1               5                   10                  15 

Leu Ile Leu Leu Phe His Lys Lys 
            20 

 
           
           
             
               16 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.103”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              91 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Trp Lys Arg Phe Leu Lys Lys 
1               5                   10                  15 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.104”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              92 

Lys Ser Lys Val Gly Trp Leu Ile Ser Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.106”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              93 

Lys Ser Lys Val Gly Trp Leu Ile Thr Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.107”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              94 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe Trp Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.108”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              95 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Trp 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.113”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              96 

Lys Ser Lys Val Gly Trp Leu Ile Gln Phe Phe His Lys Lys 
1               5                   10 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.114”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              97 

Lys Trp Gln Leu Arg Ser Lys Gly Lys Ile Lys Ile Phe Lys Ala 
1               5                   10                  15 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.115 reduced”
 
             
              98 

Met Asn Ser Gln Val Cys Glu Lys 
1               5 

 
           
           
             
               11 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.117”
 
             
              99 

Gly His Tyr Ser Phe Tyr Ser Met Asp Ile Arg 
1               5                   10 

 
           
           
             
               18 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.118”
 
             
              100 

Glu Phe Gln Leu Pro Ser Ser Gln Ile Ser Met Val Pro Asn Val Gln 
1               5                   10                  15 

Leu Lys 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.120”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              101 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Lys Leu Lys 
1               5                   10                  15 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.124”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              102 

Lys Ser Lys Val Lys Trp Leu Ile Gln Leu Trp His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.125”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              103 

Lys Ser Lys Val Gly Trp Leu Ile Tyr Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.127”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              104 

Lys Ser Lys Val Gly Phe Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.135”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              105 

Lys Ser Lys Val Gly Lys Leu Ile Gln Leu Pro His Lys Lys 
1               5                   10 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.136”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              106 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Glu Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               16 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.137 reduced”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              107 

Cys Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys Cys 
1               5                   10                  15 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.138”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              108 

Lys Ser Lys Val Lys Phe Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.139”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              109 

Lys Ser Lys Val Gly Tyr Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.141”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              110 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Trp Leu Lys 
1               5                   10                  15 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.142”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              111 

Lys Ser Lys Val Gly Trp Leu Ile Gln Trp Phe His Lys Lys 
1               5                   10 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.147”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              112 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Glu Lys Lys Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               24 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.149”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              113 

Lys Trp Lys Val Phe Lys Lys Ile Glu Lys Lys Ser Lys Val Gly Trp 
1               5                   10                  15 

Leu Ile Gln Leu Phe His Lys Lys 
            20 

 
           
           
             
               20 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.150”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              114 

Lys Trp Ala Phe Ala Lys Lys Gln Lys Lys Arg Leu Lys Arg Gln Trp 
1               5                   10                  15 

Leu Lys Lys Phe 
            20 

 
           
           
             
               26 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.151”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              115 

Gln Lys Arg Phe Leu Lys Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
1               5                   10                  15 

Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
            20                  25 

 
           
           
             
               25 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.152”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              116 

Lys Arg Phe Leu Lys Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Lys 
1               5                   10                  15 

Trp Lys Ala Gln Lys Arg Phe Leu Lys 
            20                  25 

 
           
           
             
               30 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.153”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              117 

Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Lys Trp Lys Ala Gln Lys 
1               5                   10                  15 

Arg Phe Leu Lys Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
            20                  25                  30 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.161”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              118 

Lys Ser Lys Val Lys Ala Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               24 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.162”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              119 

Lys Trp Lys Ala Gln Trp Arg Phe Leu Lys Lys Ser Lys Val Gly Trp 
1               5                   10                  15 

Leu Ile Gln Leu Phe His Lys Lys 
            20 

 
           
           
             
               20 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.163”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              120 

Lys Trp Lys Ala Gln Trp Arg Phe Leu Lys Lys Trp Lys Ala Gln Trp 
1               5                   10                  15 

Arg Phe Leu Lys 
            20 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.166”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              121 

Lys Ser Lys Val Gly Val Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.167”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              122 

Lys Trp Lys Ala Gln Lys Arg Phe 
1               5 

 
           
           
             
               14 amino acids  
               amino acid  
               circular  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.168 reduced”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              123 

Cys Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Met Ser Cys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               circular  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.169 reduced”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              124 

Cys Lys Trp Lys Ala Gln Lys Arg Phe Cys 
1               5                   10 

 
           
           
             
               12 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.170”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              125 

Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Met Ser 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.241”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              126 

Lys Ser Lys Val Gly Trp Leu Ile Leu Leu Trp His Lys Lys 
1               5                   10 

 
           
           
             
               24 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.245”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              127 

Lys Trp Lys Ala Gln Phe Arg Phe Leu Lys Lys Ser Lys Val Gly Trp 
1               5                   10                  15 

Leu Ile Gln Leu Trp His Lys Lys 
            20 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.249”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              128 

Lys Ser Lys Val Gly Gly Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.250”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              129 

Lys Ser Lys Val Gly Leu Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.251”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              130 

Lys Ser Lys Val Gly Ile Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.262”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              131 

Lys Ser Lys Val Gly Trp Leu Ile Asn Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.263”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              132 

Lys Ser Lys Val Gly Trp Leu Ile Glu Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.264”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              133 

Lys Ser Lys Val Gly Trp Leu Ile Asp Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.265”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              134 

Lys Ser Lys Val Gly Trp Leu Ile Lys Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.266”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              135 

Lys Ser Lys Val Lys Val Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.267”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              136 

Lys Ser Lys Val Lys Trp Ala Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.268”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              137 

Lys Ser Lys Val Gly Val Ala Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.269”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              138 

Lys Ser Lys Val Lys Val Ala Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               28 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.270”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              139 

Lys Ser Lys Val Gly Trp Leu Ile Leu Leu Phe His Lys Lys Lys Ser 
1               5                   10                  15 

Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
            20                  25 

 
           
           
             
               28 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.271”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              140 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys Lys Ser 
1               5                   10                  15 

Lys Val Gly Trp Leu Ile Leu Leu Phe His Lys Lys 
            20                  25 

 
           
           
             
               28 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.272”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              141 

Lys Ser Lys Val Gly Trp Leu Ile Leu Leu Phe His Lys Lys Lys Ser 
1               5                   10                  15 

Lys Val Gly Trp Leu Ile Leu Leu Phe His Lys Lys 
            20                  25 

 
           
           
             
               28 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.273”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              142 

Lys Ser Lys Val Gly Trp Leu Ile Phe Leu Phe His Lys Lys Lys Ser 
1               5                   10                  15 

Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
            20                  25 

 
           
           
             
               28 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.274”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              143 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys Lys Ser 
1               5                   10                  15 

Lys Val Gly Trp Leu Ile Phe Leu Phe His Lys Lys 
            20                  25 

 
           
           
             
               28 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.275”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              144 

Lys Ser Lys Val Gly Trp Leu Ile Phe Leu Phe His Lys Lys Lys Ser 
1               5                   10                  15 

Lys Val Gly Trp Leu Ile Phe Leu Phe His Lys Lys 
            20                  25 

 
           
           
             
               24 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.276”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              145 

Lys Trp Lys Ala Gln Phe Arg Phe Leu Lys Lys Ser Lys Val Gly Trp 
1               5                   10                  15 

Leu Ile Phe Leu Phe His Lys Lys 
            20 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.278”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              146 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Ala Trp Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.280”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              147 

Ile Lys Ile Ser Gly Lys Trp Lys Ala Ala Phe Arg Phe Leu Lys 
1               5                   10                  15 

 
           
           
             
               20 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.282”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              148 

Lys Trp Lys Ala Phe Phe Arg Phe Leu Lys Lys Trp Lys Ala Phe Phe 
1               5                   10                  15 

Arg Phe Leu Lys 
            20 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.283”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              149 

Lys Ser Lys Val Lys Phe Leu Ile Lys Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               13 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.284”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              150 

Ser Lys Val Lys Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               12 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.285”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              151 

Ser Lys Val Lys Trp Leu Ile Gln Leu Phe His Lys 
1               5                   10 

 
           
           
             
               12 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.286”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              152 

Lys Val Lys Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               11 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.287”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              153 

Ser Lys Val Lys Trp Leu Ile Gln Leu Phe His 
1               5                   10 

 
           
           
             
               11 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.288”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              154 

Lys Val Lys Trp Leu Ile Gln Leu Phe His Lys 
1               5                   10 

 
           
           
             
               11 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.289”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              155 

Val Lys Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.290”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              156 

Ser Lys Val Lys Trp Leu Ile Gln Leu Phe 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.291”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              157 

Lys Val Lys Trp Leu Ile Gln Leu Phe His 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.292”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              158 

Val Lys Trp Leu Ile Gln Leu Phe His Lys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.293”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              159 

Lys Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.294”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              160 

Ser Lys Val Lys Trp Leu Ile Gln Leu 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.295”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              161 

Lys Val Lys Trp Leu Ile Gln Leu Phe 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.296”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              162 

Val Lys Trp Leu Ile Gln Leu Phe His 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.297”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              163 

Lys Trp Leu Ile Gln Leu Phe His Lys 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.298”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              164 

Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.299”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              165 

Ser Lys Val Lys Trp Leu Ile Gln 
1               5 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.300”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              166 

Lys Val Lys Trp Leu Ile Gln Leu 
1               5 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.301”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              167 

Val Lys Trp Leu Ile Gln Leu Phe 
1               5 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.302”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              168 

Lys Trp Leu Ile Gln Leu Phe His 
1               5 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.303”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              169 

Trp Leu Ile Gln Leu Phe His Lys 
1               5 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.304”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              170 

Leu Ile Gln Leu Phe His Lys Lys 
1               5 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.305”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              171 

Ser Lys Val Lys Trp Leu Ile Gln 
1               5 

 
           
           
             
               7 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.306”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              172 

Ser Lys Val Lys Trp Leu Ile 
1               5 

 
           
           
             
               7 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.307”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              173 

Val Lys Trp Leu Ile Gln Leu 
1               5 

 
           
           
             
               7 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.308”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              174 

Lys Trp Leu Ile Gln Leu Phe 
1               5 

 
           
           
             
               7 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.309”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              175 

Trp Leu Ile Gln Leu Phe His 
1               5 

 
           
           
             
               7 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.310”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              176 

Leu Ile Gln Leu Phe His Lys 
1               5 

 
           
           
             
               7 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.311”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              177 

Ile Gln Leu Phe His Lys Lys 
1               5 

 
           
           
             
               6 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.312”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              178 

Ser Lys Val Lys Trp Leu 
1               5 

 
           
           
             
               6 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.313”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              179 

Lys Val Lys Trp Leu Ile 
1               5 

 
           
           
             
               6 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.314”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              180 

Val Lys Trp Leu Ile Gln 
1               5 

 
           
           
             
               6 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.315”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              181 

Lys Trp Leu Ile Gln Leu 
1               5 

 
           
           
             
               6 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.316”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              182 

Trp Leu Ile Gln Leu Phe 
1               5 

 
           
           
             
               6 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.317”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              183 

Trp Leu Ile Gln Leu Phe 
1               5 

 
           
           
             
               6 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.318”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              184 

Ile Gln Leu Phe His Lys 
1               5 

 
           
           
             
               6 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.319”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              185 

Gln Leu Phe His Lys Lys 
1               5 

 
           
           
             
               5 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.320”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              186 

Trp Leu Ile Gln Leu 
1               5 

 
           
           
             
               6 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.321”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              187 

Trp Leu Ile Gln Leu Lys 
1               5 

 
           
           
             
               7 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.322”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              188 

Trp Leu Ile Gln Leu Lys Lys 
1               5 

 
           
           
             
               7 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.323”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              189 

Lys Trp Leu Ile Gln Leu Lys 
1               5 

 
           
           
             
               7 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.324”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              190 

Lys Trp Leu Ile Gln Leu Lys 
1               5 

 
           
           
             
               7 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.325”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              191 

Lys Lys Trp Leu Ile Gln Leu 
1               5 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.326”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              192 

Lys Lys Trp Leu Ile Gln Leu Lys 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.327”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              193 

Lys Lys Trp Leu Ile Gln Leu Lys Lys 
1               5 

 
           
           
             
               31 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.328”
 
             
              194 

Pro Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys 
 1               5                   10                  15 

Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys Ile 
            20                  25                  30 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.329”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              195 

Lys Lys Val Val Gln Val Val Lys Lys 
1               5 

 
           
           
             
               4 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.330”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              196 

Trp Leu Ile Gln 
1 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.331”
 
             
             
               Modified-site  
                1  
                /label= Acetylated 
               /note= “Position 1 is acetylated.”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              197 

Lys Lys Trp Leu Ile Gln Leu Lys Lys 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.335”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              198 

Pro Lys Trp Leu Ile Gln Leu Lys Lys 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.336”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              199 

Arg Arg Trp Leu Ile Gln Leu Arg Arg 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.337”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              200 

His His Trp Leu Ile Gln Leu His His 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.343”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              201 

Lys Lys Val Leu Ile Gln Leu Lys Lys 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.344”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              202 

Lys Lys Trp Ala Ile Gln Leu Lys Lys 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.345”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              203 

Lys Lys Trp Leu Ile Gln Ala Lys Lys 
1               5 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.348”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              204 

Lys Lys Lys Trp Leu Ile Gln Leu Lys Lys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.349”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              205 

Lys Lys Trp Leu Ile Gln Leu Lys Lys Lys 
1               5                   10 

 
           
           
             
               11 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.350”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              206 

Lys Lys Lys Trp Leu Ile Gln Leu Lys Lys Lys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.351”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              207 

Lys Lys Trp Leu Ile Gln Leu Phe Lys Lys 
1               5                   10 

 
           
           
             
               11 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.352”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              208 

Lys Lys Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.353”
 
             
              209 

Pro Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.354”
 
             
             
               Modified-site  
                1  
                /label= Acetylated 
               /note= “Position 1 is acetylated.”
 
             
              210 

Pro Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.355”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              211 

Pro Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.356”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
             
               Modified-site  
                1  
                /label= Acetylated 
               /note= “Position 1 is acetylated.”
 
             
              212 

Pro Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.357”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              213 

Lys Trp Leu Ile Gln Leu Phe His Lys Pro 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.358”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              214 

Lys Lys Trp Leu Ile Gln Leu Phe His Pro 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.373”
 
             
             
               Modified-site  
                1  
                /label= Acetylated 
               /note= “Position 1 is acetylated.”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              215 

Lys Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.377”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              216 

Lys Lys Lys Trp Ala Ile Gln Leu Lys Lys 
1               5                   10 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.378”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              217 

Pro Trp Ala Ile Gln Leu Lys Lys 
1               5 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.379”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              218 

Lys Lys Pro Trp Ala Ile Gln Leu Lys Lys 
1               5                   10 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.380”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              219 

Lys Lys Gln Leu Leu Leu Leu Lys Lys 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.381”
 
             
             
               Modified-site  
                /label= Amidation 
               /note= “The C-Terminus is Amidated.”
 
             
              220 

Lys Lys Leu Gln Leu Leu Leu Lys Lys 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                “XMP.382 
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              221 

Lys Lys Leu Leu Gln Leu Leu Lys Lys 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.383“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              222 

Lys Lys Leu Leu Leu Gln Leu Lys Lys 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.384“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              223 

Lys Lys Leu Leu Leu Leu Gln Lys Lys 
1               5 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.385“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              224 

Lys Lys Leu Leu Leu Leu Leu Lys Lys 
1               5 

 
           
           
             
               11 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.386“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              225 

Lys Trp Ala Ile Gln Leu Phe His Lys Lys Ile 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.387“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              226 

Pro Trp Ala Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               10 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.388“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              227 

Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               11 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.389“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              228 

Lys Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               11 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.390“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              229 

Lys Pro Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               12 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.391“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              230 

Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               12 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.392“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              231 

Lys Val Pro Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.393“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              232 

Lys Ser Lys Val Pro Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.399“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              233 

Lys Ser Lys Val Gly Trp Leu Ile Phe Leu Trp His Lys Lys 
1               5                   10 

 
           
           
             
               15 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.406“
 
             
              234 

Pro Lys Ser Lys Val Gly Ala Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10                  15 

 
           
           
             
               16 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.407“
 
             
              235 

Pro Lys Ser Lys Val Gly Ala Leu Ile Gln Leu Phe His Lys Lys Asp 
1               5                   10                  15 

 
           
           
             
               9 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.409“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              236 

Ser Lys Trp Ala Ile Gln Leu Lys Lys 
1               5 

 
           
           
             
               8 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.412“
 
             
              237 

Leu Lys Lys Lys Trp Ala Ile Gln 
1               5 

 
           
           
             
               19 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.413“
 
             
              238 

Lys Trp Lys Ala Gln Lys Arg Leu Lys Lys Trp Lys Ala Ala Ala Arg 
1               5                   10                  15 

Phe Leu Lys 

 
           
           
             
               14 amino acids  
               amino acid  
               linear  
             
             
               peptide  
             
             
               misc_feature  
                ”XMP.418“
 
             
             
               Modified-site  
                /label= Amidation 
               /note= ”The C-Terminus is Amidated.“
 
             
              239 

Lys Ser Lys Val Pro Trp Leu Ile Gln Leu Phe His Lys Lys 
1               5                   10 

 
           
           
             
               78 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               DNA  
             
              240 

GCTATCTGCG CATTGGATCC GATCAAAATC TCGGGTAAAT GGAAGGCCCA GAAACGCTTT     60 

CTGAAAAAGT CGAAAGTG                                                   78 

 
           
           
             
               75 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               DNA  
             
              241 

GCGGGCTCTC GAGCTTTAAA TCTTTTTATG AAACAGCTGG ATCAGCCAAC CCACTTTCGA     60 

CTTTTTCAGA AAGCG                                                      75 

 
           
           
             
               54 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               DNA  
             
              242 

GATCCGAAGT CTAAAGTGGG GKYCCTGATC CAGCTGTTCC ACAAAAAGTA AAGC           54 

 
           
           
             
               54 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               DNA  
             
              243 

TCGAGTCTTA CTTTTTGTGA AGCAGCTGGA TCAGGRMCCC CACTTTAGAC TTCG           54 

 
           
           
             
               47 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               DNA  
             
              244 

ACTTGGGCCC CTACCTTGGA TTTTGGGTCC TTTTTGTGGA ACAGCTG                   47 

 
           
           
             
               20 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               DNA  
             
              245 

TGGAACGATA AATGCCCATG                                                 20 

 
           
           
             
               813 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
             
               misc_feature  
                ”gelonin“
 
             
              246 

GGGCTAGATA CCGTGTCATT CTCAACCAAA GGTGCCACTT ATATTACCTA CGTGAATTTC     60 

TTGAATGAGC TACGAGTTAA ATTGAAACCC GAAGGTAACA GCCATGGAAT CCCATTGCT     120 

CGCAAAAAAT GTGATGATCC TGGAAAGTGT TTCGTTTTGG TAGCGCTTTC AAATGACAA     180 

GGACAGTTGG CGGAAATAGC TATAGATGTT ACAAGTGTTT ATGTGGTGGG CTATCAAGT     240 

AGAAACAGAT CTTACTTCTT TAAAGATGCT CCAGATGCTG CTTACGAAGG CCTCTTCAA     300 

AACACAATTA AAACAAGACT TCATTTTGGC GGCAGCTATC CCTCGCTGGA AGGTGAGAA     360 

GCATATAGAG AGACAACAGA CTTGGGCATT GAACCATTAA GGATTGGCAT CAAGAAACT     420 

GATGAAAATG CGATAGACAA TTATAAACCA ACGGAGATAG CTAGTTCTCT ATTGGTTGT     480 

ATTCAAATGG TGTCTGAAGC AGCTCGATTC ACCTTTATTG AGAACCAAAT TAGAAATAA     540 

TTTCAACAGA GAATTCGCCC GGCGAATAAT ACAATCAGCC TTGAGAATAA ATGGGGTAA     600 

CTCTCGTTCC AGATCCGGAC ATCAGGTGCA AATGGAATGT TTTCGGAGGC AGTTGAATT     660 

GAACGTGCAA ATGGCAAAAA ATACTATGTC ACCGCAGTTG ATCAAGTAAA ACCCAAAAT     720 

GCACTCTTGA AGTTCGTCGA TAAAGATCCT AAAACGAGCC TTGCTGCTGA ATTGATAAT     780 

CAGAACTATG AGTCATTAGT GGGCTTTGAT TAG                                 813 

 
           
           
             
               251 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              247 

Gly Leu Asp Thr Val Ser Phe Ser Thr Lys Gly Ala Thr Tyr Ile Thr 
1               5                   10                  15 

Tyr Val Asn Phe Leu Asn Glu Leu Arg Val Lys Leu Lys Pro Glu Gln 
            20                  25                  30 

Asn Ser His Gly Ile Pro Leu Leu Arg Lys Lys Cys Asp Asp Pro Glu 
        35                  40                  45 

Lys Cys Phe Val Leu Val Ala Leu Ser Asn Asp Asn Gly Gln Leu Ala 
    50                  55                  60 

Glu Ile Ala Ile Asp Val Thr Ser Val Tyr Val Val Gly Tyr Gln Val 
65                  70                  75                  80 

Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala Pro Asp Ala Ala Tyr Gly 
                85                  90                  95 

Gly Leu Phe Lys Asn Thr Ile Lys Thr Arg Leu His Phe Gly Gly Ser 
            100                 105                 110 

Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr Arg Glu Thr Thr Asp Leu 
        115                 120                 125 

Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys Lys Leu Asp Glu Asn Ala 
    130                 135                 140 

Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala Ser Ser Leu Leu Val Val 
145                 150                 155                 160 

Ile Gln Met Val Ser Glu Ala Ala Arg Phe Thr Phe Ile Glu Asn Gln 
                165                 170                 175 

Ile Arg Asn Asn Phe Gln Gln Arg Ile Arg Pro Ala Asn Asn Thr Ile 
            180                 185                 190 

Ser Leu Glu Asn Lys Trp Gly Lys Leu Ser Phe Gln Ile Arg Thr Ser 
        195                 200                 205 

Gly Ala Asn Gly Met Phe Ser Glu Ala Val Glu Leu Glu Arg Ala Asp 
    210                 215                 220 

Gly Lys Lys Tyr Tyr Val Thr Ala Val Asp Gln Val Lys Pro Lys Ile 
225                 230                 235                 240 

Ala Leu Leu Lys Phe Val Asp Lys Asp Pro Lys 
                245                 250 

 
           
           
             
               557 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               protein  
             
             
               &lt;Unknown&gt; 
             
             
               CDS  
                66..548 

 
             
             
               misc_feature  
               residues 1-65  
                /label= EcoRI 
               /note=+38residues 1-65 comprise EcoRI site to begining of 
              pel B.“
 
             
             
               misc_feature  
               AA 1-22  
                /label= pel B 
               /note=+38pel B is the leader sequence from the pectate 
              lyase gene of Erwinia carotovora.“
 
             
             
               misc_feature  
               AA 23-161  
                /label= ”Bone D“
               /note=+38Bone D is the subunit of human osteogenic protein 
              (see, U.S. Patent No. 5,284,756 e.g., Fig. 6, Example 9, 
              Seq ID NOs 1 and 2.“
 
             
             
               misc_feature  
               residues 549-557  
                /label= XhoI 
               /note=+38residues 549-557 comprise stop codon and XhoI 
              site.“
 
             
              248 

GAATTCCTGC AGGTCTATGG AACGATAAAT GCCCATGAAA ATTCTATTTC AAGGAGACAG     60 

TCATA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA        107 
      Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu 
        1               5                  10 

CTC GCT GCC CAA CCA GCG ATG GCG TCC ACG GGG AGC AAA CAG CGC AGC      155 
Leu Ala Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser 
 15                  20                  25                  30 

CAG AAC CGC TCC AAG ACG CCC AAG AAC CAG GAA GCC CTG CGG ATG GCC      203 
Gln Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala 
                 35                  40                  45 

AAC GTG GCA GAG AAC AGC AGC AGC GAC CAG AGG CAG GCC TGT AAG AAG      251 
Asn Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys 
             50                  55                  60 

CAC GAG CTG TAT GTC AGC TTC CGA GAC CTG GGC TGG CAG GAC TGG ATC      299 
His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile 
         65                  70                  75 

ATC GCG CCT GAA GGC TAC GCC GCC TAC TAC TGT GAG GGG GAG TGT GCC      347 
Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 
     80                  85                  90 

TTC CCT CTG AAC TCC TAC ATG AAC GCC ACC AAC CAC GCC ATC GTG CAG      395 
Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln 
 95                 100                 105                 110 

ACG CTG GTC CAC TTC ATC AAC CCG GAA ACG GTG CCC AAG CCC TGC TGT      443 
Thr Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys 
                115                 120                 125 

GCG CCC ACG CAG CTC AAT GCC ATC TCC GTC CTC TAC TTC GAT GAC AGC      491 
Ala Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser 
            130                 135                 140 

TCC AAC GTC ATC CTG AAG AAA TAC AGA AAC ATG GTG GTC CGG GCC TGT      539 
Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys 
        145                 150                 155 

GGC TGC CAC TAGCTCGAG                                                557 
Gly Cys His 
    160 

 
           
           
             
               161 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              249 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
  1               5                  10                  15 

Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn 
             20                  25                  30 

Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val 
         35                  40                  45 

Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu 
     50                  55                  60 

Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala 
 65                  70                  75                  80 

Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro 
                 85                  90                  95 

Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu 
            100                 105                 110 

Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro 
        115                 120                 125 

Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn 
    130                 135                 140 

Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys 
145                 150                 155                 160 

His 

 
           
           
             
               1072 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               protein  
             
             
               CDS  
                66..1061 

 
             
             
               misc_feature  
               residues 1-65  
                /label= EcoRI 
               /note=+38residues 1-65 comprise EcoRI site to beginning of 
              pel B.“
 
             
             
               misc_feature  
               AA 1-22  
                /label= pel B 
               /note=+38pel B is the leader sequence from the pectate 
              lyase gene of Erwinia caratovora.“
 
             
             
               misc_feature  
               AA 23-273  
                /label= ”gelonin“
               /note=+38gelonin (see U.S. Patent No. 5,416,202).“
 
             
             
               misc_feature  
               AA 274-276  
                /label= EagI 
               /note=+38EagI cloning site.“
 
             
             
               misc_feature  
               AA 277-296  
                /label= SLT linker 
               /note=+38SLT from shiga-like-toxin gene.“
 
             
             
               misc_feature  
               AA 297-298  
                /label= FspI/ScaI 
               /note=+38FspI and ScaI cloning sites.“
 
             
             
               misc_feature  
               AA 299-302  
                /label= cleavage linker 
               /note=+38Ala-Leu-Asp-Pro linking sequence with Asp-Pro 
              cleavage site.“
 
             
             
               misc_feature  
               AA 303-332  
                /label= peptide sequence 
               /note=+38BPI-derived peptide.“
 
             
             
               misc_feature  
               residues 1062-1072  
                /label= XhoI 
               /note=+38residues 1062-1072 comprise stop codon and XhoI 
              site.“
 
             
              250 

GAATTCCTGC AGGTCTATGG AACGATAAAT GCCCATGAAA ATTCTATTTC AAGGAGACAG     60 

TCATA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA        107 
      Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu 
        1               5                  10 

CTC GCT GCC CAA CCA GCG ATG GCG GGC CTG GAC ACC GTG AGC TTT AGC      155 
Leu Ala Ala Gln Pro Ala Met Ala Gly Leu Asp Thr Val Ser Phe Ser 
 15                  20                  25                  30 

ACT AAA GGT GCC ACT TAT ATT ACC TAC GTG AAT TTC TTG AAT GAG CTA      203 
Thr Lys Gly Ala Thr Tyr Ile Thr Tyr Val Asn Phe Leu Asn Glu Leu 
                 35                  40                  45 

CGA GTT AAA TTG AAA CCC GAA GGT AAC AGC CAT GGA ATC CCA TTG CTG      251 
Arg Val Lys Leu Lys Pro Glu Gly Asn Ser His Gly Ile Pro Leu Leu 
             50                  55                  60 

CGC AAA AAA TGT GAT GAT CCT GGA AAG TGT TTC GTT TTG GTA GCG CTT      299 
Arg Lys Lys Cys Asp Asp Pro Gly Lys Cys Phe Val Leu Val Ala Leu 
         65                  70                  75 

TCA AAT GAC AAT GGA CAG TTG GCG GAA ATA GCT ATA GAT GTT ACA AGT      347 
Ser Asn Asp Asn Gly Gln Leu Ala Glu Ile Ala Ile Asp Val Thr Ser 
     80                  85                  90 

GTT TAT GTG GTG GGC TAT CAA GTA AGA AAC AGA TCT TAC TTC TTT AAA      395 
Val Tyr Val Val Gly Tyr Gln Val Arg Asn Arg Ser Tyr Phe Phe Lys 
 95                 100                 105                 110 

GAT GCT CCA GAT GCT GCT TAC GAA GGC CTC TTC AAA AAC ACA ATT AAA      443 
Asp Ala Pro Asp Ala Ala Tyr Glu Gly Leu Phe Lys Asn Thr Ile Lys 
                115                 120                 125 

ACA AGA CTT CAT TTT GGC GGC ACG TAT CCC TCG CTG GAA GGT GAG AAG      491 
Thr Arg Leu His Phe Gly Gly Thr Tyr Pro Ser Leu Glu Gly Glu Lys 
            130                 135                 140 

GCA TAT AGA GAG ACA ACA GAC TTG GGC ATT GAA CCA TTA AGG ATT GGC      539 
Ala Tyr Arg Glu Thr Thr Asp Leu Gly Ile Glu Pro Leu Arg Ile Gly 
        145                 150                 155 

ATC AAG AAA CTT GAT GAA AAT GCG ATA GAC AAT TAT AAA CCA ACG GAG      587 
Ile Lys Lys Leu Asp Glu Asn Ala Ile Asp Asn Tyr Lys Pro Thr Glu 
    160                 165                 170 

ATA GCT AGT TCT CTA TTG GTT GTT ATT CAA ATG GTG TCT GAA GCA GCT      635 
Ile Ala Ser Ser Leu Leu Val Val Ile Gln Met Val Ser Glu Ala Ala 
175                 180                 185                 190 

CGA TTC ACC TTT ATT GAG AAC CAA ATT AGA AAT AAC TTT CAA CAG AGA      683 
Arg Phe Thr Phe Ile Glu Asn Gln Ile Arg Asn Asn Phe Gln Gln Arg 
                195                 200                 205 

ATT CGC CCG GCG AAT AAT ACA ATC AGC CTT GAG AAT AAA TGG GGT AAA      731 
Ile Arg Pro Ala Asn Asn Thr Ile Ser Leu Glu Asn Lys Trp Gly Lys 
            210                 215                 220 

CTC TCG TTC CAG ATC CGG ACA TCA GGT GCA AAT GGA ATG TTT TCG GAG      779 
Leu Ser Phe Gln Ile Arg Thr Ser Gly Ala Asn Gly Met Phe Ser Glu 
        225                 230                 235 

GCA GTT GAA TTG GAA CGT GCA AAT GGC AAA AAA TAC TAT GTC ACC GCA      827 
Ala Val Glu Leu Glu Arg Ala Asn Gly Lys Lys Tyr Tyr Val Thr Ala 
    240                 245                 250 

GTT GAT CAA GTA AAA CCC AAA ATA GCA CTC TTG AAG TTC GTC GAT AAA      875 
Val Asp Gln Val Lys Pro Lys Ile Ala Leu Leu Lys Phe Val Asp Lys 
255                 260                 265                 270 

GAT CCT AAA TCG GCC GCA TGT CAT CAT CAT GCA TCG CGA GTT GCC AGA      923 
Asp Pro Lys Ser Ala Ala Cys His His His Ala Ser Arg Val Ala Arg 
                275                 280                 285 

ATG GCA TCT GAT GAG TTT CCT TCT ATG TGC GCA ATG GCA TTG GAT CCG      971 
Met Ala Ser Asp Glu Phe Pro Ser Met Cys Ala Met Ala Leu Asp Pro 
            290                 295                 300 

ATC AAA ATC TCG GGT AAA TGG AAG GCC CAG AAA CGC TTT CTG AAA AAG     1019 
Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Lys 
        305                 310                 315 

TCG AAA GTG GGT TGG CTG ATC CAG CTG TTT CAT AAA AAG ATT             1061 
Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys Ile 
    320                 325                 330 

TAAAGCTCGA G                                                        1072 

 
           
           
             
               332 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              251 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
  1               5                  10                  15 

Ala Gln Pro Ala Met Ala Gly Leu Asp Thr Val Ser Phe Ser Thr Lys 
             20                  25                  30 

Gly Ala Thr Tyr Ile Thr Tyr Val Asn Phe Leu Asn Glu Leu Arg Val 
         35                  40                  45 

Lys Leu Lys Pro Glu Gly Asn Ser His Gly Ile Pro Leu Leu Arg Lys 
     50                  55                  60 

Lys Cys Asp Asp Pro Gly Lys Cys Phe Val Leu Val Ala Leu Ser Asn 
 65                  70                  75                  80 

Asp Asn Gly Gln Leu Ala Glu Ile Ala Ile Asp Val Thr Ser Val Tyr 
                 85                  90                  95 

Val Val Gly Tyr Gln Val Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala 
            100                 105                 110 

Pro Asp Ala Ala Tyr Glu Gly Leu Phe Lys Asn Thr Ile Lys Thr Arg 
        115                 120                 125 

Leu His Phe Gly Gly Thr Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr 
    130                 135                 140 

Arg Glu Thr Thr Asp Leu Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys 
145                 150                 155                 160 

Lys Leu Asp Glu Asn Ala Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala 
                165                 170                 175 

Ser Ser Leu Leu Val Val Ile Gln Met Val Ser Glu Ala Ala Arg Phe 
            180                 185                 190 

Thr Phe Ile Glu Asn Gln Ile Arg Asn Asn Phe Gln Gln Arg Ile Arg 
        195                 200                 205 

Pro Ala Asn Asn Thr Ile Ser Leu Glu Asn Lys Trp Gly Lys Leu Ser 
    210                 215                 220 

Phe Gln Ile Arg Thr Ser Gly Ala Asn Gly Met Phe Ser Glu Ala Val 
225                 230                 235                 240 

Glu Leu Glu Arg Ala Asn Gly Lys Lys Tyr Tyr Val Thr Ala Val Asp 
                245                 250                 255 

Gln Val Lys Pro Lys Ile Ala Leu Leu Lys Phe Val Asp Lys Asp Pro 
            260                 265                 270 

Lys Ser Ala Ala Cys His His His Ala Ser Arg Val Ala Arg Met Ala 
        275                 280                 285 

Ser Asp Glu Phe Pro Ser Met Cys Ala Met Ala Leu Asp Pro Ile Lys 
    290                 295                 300 

Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Lys Ser Lys 
305                 310                 315                 320 

Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys Ile 
                325                 330 

 
           
           
             
               1003 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               protein  
             
             
               CDS  
                66..992 

 
             
             
               misc_feature  
               residues 1-65  
                /label= EcoRI 
               /note=+38residues 1-65 comprise EcoRI site to beginning of 
              pel B.“
 
             
             
               misc_feature  
               AA 1-22  
                /label= pel B 
              /note=”pel B is the leader sequence from the pectate lyase 
              gene of Erwinia caratovora.“
 
             
             
               misc_feature  
               AA 23-273  
                /label= ”gelonin“
               /note=+38gelonin (see U.S. Patent No. 5,416,202).“
 
             
             
               misc_feature  
               AA 274-275  
                /label= EagI 
               /note=+38EagI cloning site.“
 
             
             
               misc_feature  
               AA 276-279  
                /label= cleavage linker 
               /note=+38Ala-Leu-Asp-Pro linking sequence with Asp-Pro 
               cleavage site.“
 
             
             
               misc_feature  
               AA 280-309  
                /label= peptide sequence 
               /note=+38BPI-derived peptide.“
 
             
             
               misc_feature  
               residues 993-1011  
                /label= XhoI 
               /note=+38residues 993-1003 comprise stop codon and XhoI 
               site.“
 
             
              252 

GAATTCCTGC AGGTCTATGG AACGATAAAT GCCCATGAAA ATTCTATTTC AAGGAGACAG     60 

TCATA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA        107 
      Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu 
        1               5                  10 

CTC GCT GCC CAA CCA GCG ATG GCG GGC CTG GAC ACC GTG AGC TTT AGC      155 
Leu Ala Ala Gln Pro Ala Met Ala Gly Leu Asp Thr Val Ser Phe Ser 
 15                  20                  25                  30 

ACT AAA GGT GCC ACT TAT ATT ACC TAC GTG AAT TTC TTG AAT GAG CTA      203 
Thr Lys Gly Ala Thr Tyr Ile Thr Tyr Val Asn Phe Leu Asn Glu Leu 
                 35                  40                  45 

CGA GTT AAA TTG AAA CCC GAA GGT AAC AGC CAT GGA ATC CCA TTG CTG      251 
Arg Val Lys Leu Lys Pro Glu Gly Asn Ser His Gly Ile Pro Leu Leu 
             50                  55                  60 

CGC AAA AAA TGT GAT GAT CCT GGA AAG TGT TTC GTT TTG GTA GCG CTT      299 
Arg Lys Lys Cys Asp Asp Pro Gly Lys Cys Phe Val Leu Val Ala Leu 
         65                  70                  75 

TCA AAT GAC AAT GGA CAG TTG GCG GAA ATA GCT ATA GAT GTT ACA AGT      347 
Ser Asn Asp Asn Gly Gln Leu Ala Glu Ile Ala Ile Asp Val Thr Ser 
     80                  85                  90 

GTT TAT GTG GTG GGC TAT CAA GTA AGA AAC AGA TCT TAC TTC TTT AAA      395 
Val Tyr Val Val Gly Tyr Gln Val Arg Asn Arg Ser Tyr Phe Phe Lys 
 95                 100                 105                 110 

GAT GCT CCA GAT GCT GCT TAC GAA GGC CTC TTC AAA AAC ACA ATT AAA      443 
Asp Ala Pro Asp Ala Ala Tyr Glu Gly Leu Phe Lys Asn Thr Ile Lys 
                115                 120                 125 

ACA AGA CTT CAT TTT GGC GGC ACG TAT CCC TCG CTG GAA GGT GAG AAG      491 
Thr Arg Leu His Phe Gly Gly Thr Tyr Pro Ser Leu Glu Gly Glu Lys 
            130                 135                 140 

GCA TAT AGA GAG ACA ACA GAC TTG GGC ATT GAA CCA TTA AGG ATT GGC      539 
Ala Tyr Arg Glu Thr Thr Asp Leu Gly Ile Glu Pro Leu Arg Ile Gly 
        145                 150                 155 

ATC AAG AAA CTT GAT GAA AAT GCG ATA GAC AAT TAT AAA CCA ACG GAG      587 
Ile Lys Lys Leu Asp Glu Asn Ala Ile Asp Asn Tyr Lys Pro Thr Glu 
    160                 165                 170 

ATA GCT AGT TCT CTA TTG GTT GTT ATT CAA ATG GTG TCT GAA GCA GCT      635 
Ile Ala Ser Ser Leu Leu Val Val Ile Gln Met Val Ser Glu Ala Ala 
175                 180                 185                 190 

CGA TTC ACC TTT ATT GAG AAC CAA ATT AGA AAT AAC TTT CAA CAG AGA      683 
Arg Phe Thr Phe Ile Glu Asn Gln Ile Arg Asn Asn Phe Gln Gln Arg 
                195                 200                 205 

ATT CGC CCG GCG AAT AAT ACA ATC AGC CTT GAG AAT AAA TGG GGT AAA      731 
Ile Arg Pro Ala Asn Asn Thr Ile Ser Leu Glu Asn Lys Trp Gly Lys 
            210                 215                 220 

CTC TCG TTC CAG ATC CGG ACA TCA GGT GCA AAT GGA ATG TTT TCG GAG      779 
Leu Ser Phe Gln Ile Arg Thr Ser Gly Ala Asn Gly Met Phe Ser Glu 
        225                 230                 235 

GCA GTT GAA TTG GAA CGT GCA AAT GGC AAA AAA TAC TAT GTC ACC GCA      827 
Ala Val Glu Leu Glu Arg Ala Asn Gly Lys Lys Tyr Tyr Val Thr Ala 
    240                 245                 250 

GTT GAT CAA GTA AAA CCC AAA ATA GCA CTC TTG AAG TTC GTC GAT AAA      875 
Val Asp Gln Val Lys Pro Lys Ile Ala Leu Leu Lys Phe Val Asp Lys 
255                 260                 265                 270 

GAT CCT AAA TCG GCC GCA TTG GAT CCG ATC AAA ATC TCG GGT AAA TGG      923 
Asp Pro Lys Ser Ala Ala Leu Asp Pro Ile Lys Ile Ser Gly Lys Trp 
                275                 280                 285 

AAG GCC CAG AAA CGC TTT CTG AAA AAG TCG AAA GTG GGT TGG CTG ATC      971 
Lys Ala Gln Lys Arg Phe Leu Lys Lys Ser Lys Val Gly Trp Leu Ile 
            290                 295                 300 

CAG CTG TTT CAT AAA AAG ATT TAAAGCTCGA G                            1003 
Gln Leu Phe His Lys Lys Ile 
        305 

 
           
           
             
               309 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              253 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
  1               5                  10                  15 

Ala Gln Pro Ala Met Ala Gly Leu Asp Thr Val Ser Phe Ser Thr Lys 
             20                  25                  30 

Gly Ala Thr Tyr Ile Thr Tyr Val Asn Phe Leu Asn Glu Leu Arg Val 
         35                  40                  45 

Lys Leu Lys Pro Glu Gly Asn Ser His Gly Ile Pro Leu Leu Arg Lys 
     50                  55                  60 

Lys Cys Asp Asp Pro Gly Lys Cys Phe Val Leu Val Ala Leu Ser Asn 
 65                  70                  75                  80 

Asp Asn Gly Gln Leu Ala Glu Ile Ala Ile Asp Val Thr Ser Val Tyr 
                 85                  90                  95 

Val Val Gly Tyr Gln Val Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala 
            100                 105                 110 

Pro Asp Ala Ala Tyr Glu Gly Leu Phe Lys Asn Thr Ile Lys Thr Arg 
        115                 120                 125 

Leu His Phe Gly Gly Thr Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr 
    130                 135                 140 

Arg Glu Thr Thr Asp Leu Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys 
145                 150                 155                 160 

Lys Leu Asp Glu Asn Ala Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala 
                165                 170                 175 

Ser Ser Leu Leu Val Val Ile Gln Met Val Ser Glu Ala Ala Arg Phe 
            180                 185                 190 

Thr Phe Ile Glu Asn Gln Ile Arg Asn Asn Phe Gln Gln Arg Ile Arg 
        195                 200                 205 

Pro Ala Asn Asn Thr Ile Ser Leu Glu Asn Lys Trp Gly Lys Leu Ser 
    210                 215                 220 

Phe Gln Ile Arg Thr Ser Gly Ala Asn Gly Met Phe Ser Glu Ala Val 
225                 230                 235                 240 

Glu Leu Glu Arg Ala Asn Gly Lys Lys Tyr Tyr Val Thr Ala Val Asp 
                245                 250                 255 

Gln Val Lys Pro Lys Ile Ala Leu Leu Lys Phe Val Asp Lys Asp Pro 
            260                 265                 270 

Lys Ser Ala Ala Leu Asp Pro Ile Lys Ile Ser Gly Lys Trp Lys Ala 
        275                 280                 285 

Gln Lys Arg Phe Leu Lys Lys Ser Lys Val Gly Trp Leu Ile Gln Leu 
    290                 295                 300 

Phe His Lys Lys Ile 
305 

 
           
           
             
               658 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               protein  
             
             
               CDS  
                66..647 

 
             
             
               misc_feature  
               residues 1-65  
                /label= EcoRI 
               /note=+38residues 1-65 comprise EcoRI site to beginning of 
               pel B.“
 
             
             
               misc_feature  
               AA 1-22  
                /label= pel B 
               /note=+38pel B is the leader sequence from the pectate 
               lyase gene of Erwinia caratovora.“
 
             
             
               misc_feature  
               AA 23-161  
                /label= ”Bone D“
               /note=+38Bone D is the subunit of human osteogenic protein 
               (see, U.S. Patent No. 5,284,756 e.g., Fig. 6, Example 9, 
               Seq ID NOs 1 and 2.“
 
             
             
               misc_feature  
               AA 162-165  
                /label= cleavage linker 
               /note=+38Ala-Leu-Asp-Pro linking sequence with Asp-Pro 
               cleavage site.“
 
             
             
               misc_feature  
               AA 166-194  
                /label= peptide sequence 
               /note=+38BPI-derived peptide.“
 
             
             
               misc_feature  
               residues 648-658  
                /label= XhoI 
               /note=+38residues 648-658 comprise stop codon and XhoI 
               site.“
 
             
              254 

GAATTCCTGC AGGTCTATGG AACGATAAAT GCCCATGAAA ATTCTATTTC AAGGAGACAG     60 

TCATA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA        107 
      Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu 
        1               5                  10 

CTC GCT GCC CAA CCA GCG ATG GCG TCC ACG GGG AGC AAA CAG CGC AGC      155 
Leu Ala Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser 
 15                  20                  25                  30 

CAG AAC CGC TCC AAG ACG CCC AAG AAC CAG GAA GCC CTG CGG ATG GCC      203 
Gln Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala 
                 35                  40                  45 

AAC GTG GCA GAG AAC AGC AGC AGC GAC CAG AGG CAG GCC TGT AAG AAG      251 
Asn Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys 
             50                  55                  60 

CAC GAG CTG TAT GTC AGC TTC CGA GAC CTG GGC TGG CAG GAC TGG ATC      299 
His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile 
         65                  70                  75 

ATC GCG CCT GAA GGC TAC GCC GCC TAC TAC TGT GAG GGG GAG TGT GCC      347 
Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 
     80                  85                  90 

TTC CCT CTG AAC TCC TAC ATG AAC GCC ACC AAC CAC GCC ATC GTG CAG      395 
Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln 
 95                 100                 105                 110 

ACG CTG GTC CAC TTC ATC AAC CCG GAA ACG GTG CCC AAG CCC TGC TGT      443 
Thr Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys 
                115                 120                 125 

GCG CCC ACG CAG CTC AAT GCC ATC TCC GTC CTC TAC TTC GAT GAC AGC      491 
Ala Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser 
            130                 135                 140 

TCC AAC GTC ATC CTG AAG AAA TAC AGA AAC ATG GTG GTC CGG GCC TGT      539 
Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys 
        145                 150                 155 

GGC TGC CAC GCA TTG GAT CCG ATC AAA ATC TCG GGT AAA TGG AAG GCC      587 
Gly Cys His Ala Leu Asp Pro Ile Lys Ile Ser Gly Lys Trp Lys Ala 
    160                 165                 170 

CAG AAA CGC TTT CTG AAA AAG TCG AAA GTG GGT TGG CTG ATC CAG CTG      635 
Gln Lys Arg Phe Leu Lys Lys Ser Lys Val Gly Trp Leu Ile Gln Leu 
175                 180                 185                 190 

TTT CAT AAA AAG ATTAGCTCGA G                                         658 
Phe His Lys Lys 

 
           
           
             
               194 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              255 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
  1               5                  10                  15 

Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn 
             20                  25                  30 

Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val 
         35                  40                  45 

Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu 
     50                  55                  60 

Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala 
 65                  70                  75                  80 

Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro 
                 85                  90                  95 

Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu 
            100                 105                 110 

Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro 
        115                 120                 125 

Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn 
    130                 135                 140 

Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys 
145                 150                 155                 160 

His Ala Leu Asp Pro Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys 
                165                 170                 175 

Arg Phe Leu Lys Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His 
            180                 185                 190 

Lys Lys 

 
           
           
             
               613 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               protein  
             
             
               CDS  
                66..602 

 
             
             
               misc_feature  
               residues 1-65  
                /label= EcoRI 
               /note=+38residues 1-65 comprise EcoRI site to beginning of 
               pel B.“
 
             
             
               misc_feature  
               AA 1-22  
                /label= pel B 
               /note=+38pel B is the leader sequence from the pectate 
               lyase gene of Erwinia caratovora.“
 
             
             
               misc_feature  
               AA 23-161  
                /label= ”Bone D“
               /note=+38Bone D is the subunit of human osteogenic protein 
               (see, U.S. Patent No. 5,284,756 e.g., Fig. 6, Example 9, 
               Seq ID NOs 1 and 2.“
 
             
             
               misc_feature  
               AA 162-165  
                /label= cleavage linker 
               /note=+38Ala-Leu-Asp-Pro linking sequence with Asp-Pro 
               cleavage site.“
 
             
             
               misc_feature  
               AA 166-179  
                /label= peptide sequence 
               /note=+38BPI-derived peptide.“
 
             
             
               misc_feature  
               residues 603-613  
                /label= XhoI 
               /note=+38residues 603-613 comprise stop codon and XhoI 
               site.“
 
             
              256 

GAATTCCTGC AGGTCTATGG AACGATAAAT GCCCATGAAA ATTCTATTTC AAGGAGACAG     60 

TCATA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA        107 
      Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu 
        1               5                  10 

CTC GCT GCC CAA CCA GCG ATG GCG TCC ACG GGG AGC AAA CAG CGC AGC      155 
Leu Ala Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser 
 15                  20                  25                  30 

CAG AAC CGC TCC AAG ACG CCC AAG AAC CAG GAA GCC CTG CGG ATG GCC      203 
Gln Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala 
                 35                  40                  45 

AAC GTG GCA GAG AAC AGC AGC AGC GAC CAG AGG CAG GCC TGT AAG AAG      251 
Asn Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys 
             50                  55                  60 

CAC GAG CTG TAT GTC AGC TTC CGA GAC CTG GGC TGG CAG GAC TGG ATC      299 
His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile 
         65                  70                  75 

ATC GCG CCT GAA GGC TAC GCC GCC TAC TAC TGT GAG GGG GAG TGT GCC      347 
Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 
     80                  85                  90 

TTC CCT CTG AAC TCC TAC ATG AAC GCC ACC AAC CAC GCC ATC GTG CAG      395 
Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln 
 95                 100                 105                 110 

ACG CTG GTC CAC TTC ATC AAC CCG GAA ACG GTG CCC AAG CCC TGC TGT      443 
Thr Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys 
                115                 120                 125 

GCG CCC ACG CAG CTC AAT GCC ATC TCC GTC CTC TAC TTC GAT GAC AGC      491 
Ala Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser 
            130                 135                 140 

TCC AAC GTC ATC CTG AAG AAA TAC AGA AAC ATG GTG GTC CGG GCC TGT      539 
Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys 
        145                 150                 155 

GGC TGC CAC GCA TTG GAT CCG AAG TCT AAA GTG GGG GCC CTG ATC CAG      587 
Gly Cys His Ala Leu Asp Pro Lys Ser Lys Val Gly Ala Leu Ile Gln 
    160                 165                 170 

CTG TTC CAC AAA AAG TAAAGCTCGA G                                     613 
Leu Phe His Lys Lys 
175 

 
           
           
             
               179 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              257 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
  1               5                  10                  15 

Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn 
             20                  25                  30 

Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val 
         35                  40                  45 

Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu 
     50                  55                  60 

Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala 
 65                  70                  75                  80 

Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro 
                 85                  90                  95 

Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu 
            100                 105                 110 

Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro 
        115                 120                 125 

Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn 
    130                 135                 140 

Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys 
145                 150                 155                 160 

His Ala Leu Asp Pro Lys Ser Lys Val Gly Ala Leu Ile Gln Leu Phe 
                165                 170                 175 

His Lys Lys 

 
           
           
             
               955 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               protein  
             
             
               CDS  
                66..944 

 
             
             
               misc_feature  
               residues 1-65  
                /label= EcoRI 
               /note=+38residues 1-65 comprise EcoRI site to beginning of 
               pel B.“
 
             
             
               misc_feature  
               AA 1-22  
                /label= pel B 
               /note=+38pel B is the leader sequence from the pectate 
               lyase gene of Erwinia caratovora.“
 
             
             
               misc_feature  
               AA 23-273  
                /label= ”gelonin“
               /note=+38gelonin (see U.S. Patent No. 5,416,202).“
 
             
             
               misc_feature  
               AA 274-275  
                /label= EagI 
               /note=+38EagI cloning site.“
 
             
             
               misc_feature  
               AA 276-279  
                /label= cleavage linker 
               /note=+38Ala-Leu-Asp-Pro linking sequence with Asp-Pro 
               cleavage site.“
 
             
             
               misc_feature  
               AA 280-293  
                /label= peptide sequence 
               /note=+38BPI-derived peptide.“
 
             
             
               misc_feature  
               residues 945-954  
                /label= XhoI 
               /note=+38residues 945-955 comprise stop codon and XhoI 
               site.“
 
             
              258 

GAATTCCTGC AGGTCTATGG AACGATAAAT GCCCATGAAA ATTCTATTTC AAGGAGACAG     60 

TCATA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA        107 
      Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu 
        1               5                  10 

CTC GCT GCC CAA CCA GCG ATG GCG GGC CTG GAC ACC GTG AGC TTT AGC      155 
Leu Ala Ala Gln Pro Ala Met Ala Gly Leu Asp Thr Val Ser Phe Ser 
 15                  20                  25                  30 

ACT AAA GGT GCC ACT TAT ATT ACC TAC GTG AAT TTC TTG AAT GAG CTA      203 
Thr Lys Gly Ala Thr Tyr Ile Thr Tyr Val Asn Phe Leu Asn Glu Leu 
                 35                  40                  45 

CGA GTT AAA TTG AAA CCC GAA GGT AAC AGC CAT GGA ATC CCA TTG CTG      251 
Arg Val Lys Leu Lys Pro Glu Gly Asn Ser His Gly Ile Pro Leu Leu 
             50                  55                  60 

CGC AAA AAA TGT GAT GAT CCT GGA AAG TGT TTC GTT TTG GTA GCG CTT      299 
Arg Lys Lys Cys Asp Asp Pro Gly Lys Cys Phe Val Leu Val Ala Leu 
         65                  70                  75 

TCA AAT GAC AAT GGA CAG TTG GCG GAA ATA GCT ATA GAT GTT ACA AGT      347 
Ser Asn Asp Asn Gly Gln Leu Ala Glu Ile Ala Ile Asp Val Thr Ser 
     80                  85                  90 

GTT TAT GTG GTG GGC TAT CAA GTA AGA AAC AGA TCT TAC TTC TTT AAA      395 
Val Tyr Val Val Gly Tyr Gln Val Arg Asn Arg Ser Tyr Phe Phe Lys 
 95                 100                 105                 110 

GAT GCT CCA GAT GCT GCT TAC GAA GGC CTC TTC AAA AAC ACA ATT AAA      443 
Asp Ala Pro Asp Ala Ala Tyr Glu Gly Leu Phe Lys Asn Thr Ile Lys 
                115                 120                 125 

ACA AGA CTT CAT TTT GGC GGC ACG TAT CCC TCG CTG GAA GGT GAG AAG      491 
Thr Arg Leu His Phe Gly Gly Thr Tyr Pro Ser Leu Glu Gly Glu Lys 
            130                 135                 140 

GCA TAT AGA GAG ACA ACA GAC TTG GGC ATT GAA CCA TTA AGG ATT GGC      539 
Ala Tyr Arg Glu Thr Thr Asp Leu Gly Ile Glu Pro Leu Arg Ile Gly 
        145                 150                 155 

ATC AAG AAA CTT GAT GAA AAT GCG ATA GAC AAT TAT AAA CCA ACG GAG      587 
Ile Lys Lys Leu Asp Glu Asn Ala Ile Asp Asn Tyr Lys Pro Thr Glu 
    160                 165                 170 

ATA GCT AGT TCT CTA TTG GTT GTT ATT CAA ATG GTG TCT GAA GCA GCT      635 
Ile Ala Ser Ser Leu Leu Val Val Ile Gln Met Val Ser Glu Ala Ala 
175                 180                 185                 190 

CGA TTC ACC TTT ATT GAG AAC CAA ATT AGA AAT AAC TTT CAA CAG AGA      683 
Arg Phe Thr Phe Ile Glu Asn Gln Ile Arg Asn Asn Phe Gln Gln Arg 
                195                 200                 205 

ATT CGC CCG GCG AAT AAT ACA ATC AGC CTT GAG AAT AAA TGG GGT AAA      731 
Ile Arg Pro Ala Asn Asn Thr Ile Ser Leu Glu Asn Lys Trp Gly Lys 
            210                 215                 220 

CTC TCG TTC CAG ATC CGG ACA TCA GGT GCA AAT GGA ATG TTT TCG GAG      779 
Leu Ser Phe Gln Ile Arg Thr Ser Gly Ala Asn Gly Met Phe Ser Glu 
        225                 230                 235 

GCA GTT GAA TTG GAA CGT GCA AAT GGC AAA AAA TAC TAT GTC ACC GCA      827 
Ala Val Glu Leu Glu Arg Ala Asn Gly Lys Lys Tyr Tyr Val Thr Ala 
    240                 245                 250 

GTT GAT CAA GTA AAA CCC AAA ATA GCA CTC TTG AAG TTC GTC GAT AAA      875 
Val Asp Gln Val Lys Pro Lys Ile Ala Leu Leu Lys Phe Val Asp Lys 
255                 260                 265                 270 

GAT CCT AAA TCG GCC GCA TTG GAT CCG AAG TCT AAA GTG GGG GCC CTG      923 
Asp Pro Lys Ser Ala Ala Leu Asp Pro Lys Ser Lys Val Gly Ala Leu 
                275                 280                 285 

ATC CAG CTG TTC CAC AAA AAG TAAAGCTCGA G                             955 
Ile Gln Leu Phe His Lys Lys 
            290 

 
           
           
             
               293 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              259 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
  1               5                  10                  15 

Ala Gln Pro Ala Met Ala Gly Leu Asp Thr Val Ser Phe Ser Thr Lys 
             20                  25                  30 

Gly Ala Thr Tyr Ile Thr Tyr Val Asn Phe Leu Asn Glu Leu Arg Val 
         35                  40                  45 

Lys Leu Lys Pro Glu Gly Asn Ser His Gly Ile Pro Leu Leu Arg Lys 
     50                  55                  60 

Lys Cys Asp Asp Pro Gly Lys Cys Phe Val Leu Val Ala Leu Ser Asn 
 65                  70                  75                  80 

Asp Asn Gly Gln Leu Ala Glu Ile Ala Ile Asp Val Thr Ser Val Tyr 
                 85                  90                  95 

Val Val Gly Tyr Gln Val Arg Asn Arg Ser Tyr Phe Phe Lys Asp Ala 
            100                 105                 110 

Pro Asp Ala Ala Tyr Glu Gly Leu Phe Lys Asn Thr Ile Lys Thr Arg 
        115                 120                 125 

Leu His Phe Gly Gly Thr Tyr Pro Ser Leu Glu Gly Glu Lys Ala Tyr 
    130                 135                 140 

Arg Glu Thr Thr Asp Leu Gly Ile Glu Pro Leu Arg Ile Gly Ile Lys 
145                 150                 155                 160 

Lys Leu Asp Glu Asn Ala Ile Asp Asn Tyr Lys Pro Thr Glu Ile Ala 
                165                 170                 175 

Ser Ser Leu Leu Val Val Ile Gln Met Val Ser Glu Ala Ala Arg Phe 
            180                 185                 190 

Thr Phe Ile Glu Asn Gln Ile Arg Asn Asn Phe Gln Gln Arg Ile Arg 
        195                 200                 205 

Pro Ala Asn Asn Thr Ile Ser Leu Glu Asn Lys Trp Gly Lys Leu Ser 
    210                 215                 220 

Phe Gln Ile Arg Thr Ser Gly Ala Asn Gly Met Phe Ser Glu Ala Val 
225                 230                 235                 240 

Glu Leu Glu Arg Ala Asn Gly Lys Lys Tyr Tyr Val Thr Ala Val Asp 
                245                 250                 255 

Gln Val Lys Pro Lys Ile Ala Leu Leu Lys Phe Val Asp Lys Asp Pro 
            260                 265                 270 

Lys Ser Ala Ala Leu Asp Pro Lys Ser Lys Val Gly Ala Leu Ile Gln 
        275                 280                 285 

Leu Phe His Lys Lys 
    290 

 
           
           
             
               613 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               protein  
             
             
               CDS  
                66..602 

 
             
             
               misc_feature  
               residues 1-65  
                /label= EcoRI 
               /note=+38residues 1-65 comprise EcoRI site to beginning of 
               pel B.“
 
             
             
               misc_feature  
               AA 1-22  
                /label= pel B 
               /note=+38pel B is the leader sequence from the pectate 
               lyase gene of Erwinia caratovora.“
 
             
             
               misc_feature  
               AA 23-161  
                /label= ”Bone D“
               /note=+38Bone D is the subunit of human osteogenic protein 
               (see, U.S. Patent No. 5,284,756 e.g., Fig. 6, Example 9, 
               Seq ID NOs 1 and 2.“
 
             
             
               misc_feature  
               AA 162-165  
                /label= cleavage linker 
               /note=+38Ala-Leu-Asp-Pro linking sequence with Asp-Pro 
               cleavage site.“
 
             
             
               misc_feature  
               AA 166-179  
                /label= peptide sequence 
               /note=+38BPI-derived peptide.“
 
             
             
               misc_feature  
               residues 603-613  
                /label= XhoI 
               /note=+38residues 603-613 comprise stop codon and XhoI 
               site.“
 
             
              260 

GAATTCCTGC AGGTCTATGG AACGATAAAT GCCCATGAAA ATTCTATTTC AAGGAGACAG     60 

TCATA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA        107 
      Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu 
        1               5                  10 

CTC GCT GCC CAA CCA GCG ATG GCG TCC ACG GGG AGC AAA CAG CGC AGC      155 
Leu Ala Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser 
 15                  20                  25                  30 

CAG AAC CGC TCC AAG ACG CCC AAG AAC CAG GAA GCC CTG CGG ATG GCC      203 
Gln Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala 
                 35                  40                  45 

AAC GTG GCA GAG AAC AGC AGC AGC GAC CAG AGG CAG GCC TGT AAG AAG      251 
Asn Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys 
             50                  55                  60 

CAC GAG CTG TAT GTC AGC TTC CGA GAC CTG GGC TGG CAG GAC TGG ATC      299 
His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile 
         65                  70                  75 

ATC GCG CCT GAA GGC TAC GCC GCC TAC TAC TGT GAG GGG GAG TGT GCC      347 
Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 
     80                  85                  90 

TTC CCT CTG AAC TCC TAC ATG AAC GCC ACC AAC CAC GCC ATC GTG CAG      395 
Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln 
 95                 100                 105                 110 

ACG CTG GTC CAC TTC ATC AAC CCG GAA ACG GTG CCC AAG CCC TGC TGT      443 
Thr Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys 
                115                 120                 125 

GCG CCC ACG CAG CTC AAT GCC ATC TCC GTC CTC TAC TTC GAT GAC AGC      491 
Ala Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser 
            130                 135                 140 

TCC AAC GTC ATC CTG AAG AAA TAC AGA AAC ATG GTG GTC CGG GCC TGT      539 
Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys 
        145                 150                 155 

GGC TGC CAC GCA TTG GAT CCG AAG TCT AAA GTG GGG GCC CTG ATC CAG      587 
Gly Cys His Ala Leu Asp Pro Lys Ser Lys Val Gly Ala Leu Ile Gln 
    160                 165                 170 

CTG TTC CAC AAA AAG TAAAGCTCGA G                                     613 
Leu Phe His Lys Lys 
175 

 
           
           
             
               179 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              261 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
  1               5                  10                  15 

Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn 
             20                  25                  30 

Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val 
         35                  40                  45 

Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu 
     50                  55                  60 

Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala 
 65                  70                  75                  80 

Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro 
                 85                  90                  95 

Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu 
            100                 105                 110 

Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro 
        115                 120                 125 

Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn 
    130                 135                 140 

Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys 
145                 150                 155                 160 

His Ala Leu Asp Pro Lys Ser Lys Val Gly Ala Leu Ile Gln Leu Phe 
                165                 170                 175 

His Lys Lys 

 
           
           
             
               661 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               protein  
             
             
               CDS  
                66..650 

 
             
             
               misc_feature  
               residues 1-65  
                /label= EcoRI 
               /note=+38residues 1-65 comprise EcoRI site to beginning of 
               pel B.“
 
             
             
               misc_feature  
               AA 1-22  
                /label= pel B 
               /note=+38pel B is the leader sequence from the pectate 
               lyase gene of Erwinia caratovora.“
 
             
             
               misc_feature  
               AA 23-161  
                /label= ”Bone D“
               /note=+38Bone D is the subunit of human osteogenic protein 
               (see, U.S. Patent No. 5,284,756 e.g., Fig. 6, Example 9, 
               Seq ID NOs 1 and 2.“
 
             
             
               misc_feature  
               AA 162-165  
                /label= cleavage linker 
               /note=+38Ala-Leu-Asp-Pro linking sequence with Asp-Pro 
               cleavage site.“
 
             
             
               misc_feature  
               AA 166-195  
                /label= peptide sequence 
               /note=+38BPI-derived peptide.“
 
             
             
               misc_feature  
               residues 651-661  
                /label= XhoI 
               /note=+38residues 651-661 comprise stop codon and XhoI 
               site.“
 
             
              262 

GAATTCCTGC AGGTCTATGG AACGATAAAT GCCCATGAAA ATTCTATTTC AAGGAGACAG     60 

TCATA ATG AAA TAC CTA TTG CCT ACG GCA GCC GCT GGA TTG TTA TTA        107 
      Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu 
        1               5                  10 

CTC GCT GCC CAA CCA GCG ATG GCG TCC ACG GGG AGC AAA CAG CGC AGC      155 
Leu Ala Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser 
 15                  20                  25                  30 

CAG AAC CGC TCC AAG ACG CCC AAG AAC CAG GAA GCC CTG CGG ATG GCC      203 
Gln Asn Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala 
                 35                  40                  45 

AAC GTG GCA GAG AAC AGC AGC AGC GAC CAG AGG CAG GCC TGT AAG AAG      251 
Asn Val Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys 
             50                  55                  60 

CAC GAG CTG TAT GTC AGC TTC CGA GAC CTG GGC TGG CAG GAC TGG ATC      299 
His Glu Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile 
         65                  70                  75 

ATC GCG CCT GAA GGC TAC GCC GCC TAC TAC TGT GAG GGG GAG TGT GCC      347 
Ile Ala Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala 
     80                  85                  90 

TTC CCT CTG AAC TCC TAC ATG AAC GCC ACC AAC CAC GCC ATC GTG CAG      395 
Phe Pro Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln 
 95                 100                 105                 110 

ACG CTG GTC CAC TTC ATC AAC CCG GAA ACG GTG CCC AAG CCC TGC TGT      443 
Thr Leu Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys 
                115                 120                 125 

GCG CCC ACG CAG CTC AAT GCC ATC TCC GTC CTC TAC TTC GAT GAC AGC      491 
Ala Pro Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser 
            130                 135                 140 

TCC AAC GTC ATC CTG AAG AAA TAC AGA AAC ATG GTG GTC CGG GCC TGT      539 
Ser Asn Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys 
        145                 150                 155 

GGC TGC CAC GCA TTG GAT CCG AAG TCT AAA GTG GGG GCC CTG ATC CAG      587 
Gly Cys His Ala Leu Asp Pro Lys Ser Lys Val Gly Ala Leu Ile Gln 
    160                 165                 170 

CTG TTC CAC AAA AAG GAC CCA AAA TCC AAG GTA GGG GCC CTG ATC CAG      635 
Leu Phe His Lys Lys Asp Pro Lys Ser Lys Val Gly Ala Leu Ile Gln 
175                 180                 185                 190 

CTG TTC CAC AAA AAG TAAAGCTCGA G                                     661 
Leu Phe His Lys Lys 
                195 

 
           
           
             
               195 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              263 

Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 
  1               5                  10                  15 

Ala Gln Pro Ala Met Ala Ser Thr Gly Ser Lys Gln Arg Ser Gln Asn 
             20                  25                  30 

Arg Ser Lys Thr Pro Lys Asn Gln Glu Ala Leu Arg Met Ala Asn Val 
         35                  40                  45 

Ala Glu Asn Ser Ser Ser Asp Gln Arg Gln Ala Cys Lys Lys His Glu 
     50                  55                  60 

Leu Tyr Val Ser Phe Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala 
 65                  70                  75                  80 

Pro Glu Gly Tyr Ala Ala Tyr Tyr Cys Glu Gly Glu Cys Ala Phe Pro 
                 85                  90                  95 

Leu Asn Ser Tyr Met Asn Ala Thr Asn His Ala Ile Val Gln Thr Leu 
            100                 105                 110 

Val His Phe Ile Asn Pro Glu Thr Val Pro Lys Pro Cys Cys Ala Pro 
        115                 120                 125 

Thr Gln Leu Asn Ala Ile Ser Val Leu Tyr Phe Asp Asp Ser Ser Asn 
    130                 135                 140 

Val Ile Leu Lys Lys Tyr Arg Asn Met Val Val Arg Ala Cys Gly Cys 
145                 150                 155                 160 

His Ala Leu Asp Pro Lys Ser Lys Val Gly Ala Leu Ile Gln Leu Phe 
                165                 170                 175 

His Lys Lys Asp Pro Lys Ser Lys Val Gly Ala Leu Ile Gln Leu Phe 
            180                 185                 190 

His Lys Lys 
        195 

 
           
           
             
               1813 base pairs  
               nucleic acid  
               single  
               linear  
             
             
               cDNA  
             
             
               CDS  
                31..1491 

 
             
             
               mat_peptide  
                124..1491 

 
             
             
               misc_feature  
                ”rBPI“
 
             
              264 

CAGGCCTTGA GGTTTTGGCA GCTCTGGAGG ATG AGA GAG AAC ATG GCC AGG GGC      54 
                                 Met Arg Glu Asn Met Ala Arg Gly 
                                 -31 -30                 -25 

CCT TGC AAC GCG CCG AGA TGG GTG TCC CTG ATG GTG CTC GTC GCC ATA      102 
Pro Cys Asn Ala Pro Arg Trp Val Ser Leu Met Val Leu Val Ala Ile 
            -20                 -15                 -10 

GGC ACC GCC GTG ACA GCG GCC GTC AAC CCT GGC GTC GTG GTC AGG ATC      150 
Gly Thr Ala Val Thr Ala Ala Val Asn Pro Gly Val Val Val Arg Ile 
         -5                   1               5 

TCC CAG AAG GGC CTG GAC TAC GCC AGC CAG CAG GGG ACG GCC GCT CTG      198 
Ser Gln Lys Gly Leu Asp Tyr Ala Ser Gln Gln Gly Thr Ala Ala Leu 
 10                  15                  20                  25 

CAG AAG GAG CTG AAG AGG ATC AAG ATT CCT GAC TAC TCA GAC AGC TTT      246 
Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro Asp Tyr Ser Asp Ser Phe 
                 30                  35                  40 

AAG ATC AAG CAT CTT GGG AAG GGG CAT TAT AGC TTC TAC AGC ATG GAC      294 
Lys Ile Lys His Leu Gly Lys Gly His Tyr Ser Phe Tyr Ser Met Asp 
             45                  50                  55 

ATC CGT GAA TTC CAG CTT CCC AGT TCC CAG ATA AGC ATG GTG CCC AAT      342 
Ile Arg Glu Phe Gln Leu Pro Ser Ser Gln Ile Ser Met Val Pro Asn 
         60                  65                  70 

GTG GGC CTT AAG TTC TCC ATC AGC AAC GCC AAT ATC AAG ATC AGC GGG      390 
Val Gly Leu Lys Phe Ser Ile Ser Asn Ala Asn Ile Lys Ile Ser Gly 
     75                  80                  85 

AAA TGG AAG GCA CAA AAG AGA TTC TTA AAA ATG AGC GGC AAT TTT GAC      438 
Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Met Ser Gly Asn Phe Asp 
 90                  95                 100                 105 

CTG AGC ATA GAA GGC ATG TCC ATT TCG GCT GAT CTG AAG CTG GGC AGT      486 
Leu Ser Ile Glu Gly Met Ser Ile Ser Ala Asp Leu Lys Leu Gly Ser 
                110                 115                 120 

AAC CCC ACG TCA GGC AAG CCC ACC ATC ACC TGC TCC AGC TGC AGC AGC      534 
Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cys Ser Ser Cys Ser Ser 
            125                 130                 135 

CAC ATC AAC AGT GTC CAC GTG CAC ATC TCA AAG AGC AAA GTC GGG TGG      582 
His Ile Asn Ser Val His Val His Ile Ser Lys Ser Lys Val Gly Trp 
        140                 145                 150 

CTG ATC CAA CTC TTC CAC AAA AAA ATT GAG TCT GCG CTT CGA AAC AAG      630 
Leu Ile Gln Leu Phe His Lys Lys Ile Glu Ser Ala Leu Arg Asn Lys 
    155                 160                 165 

ATG AAC AGC CAG GTC TGC GAG AAA GTG ACC AAT TCT GTA TCC TCC AAG      678 
Met Asn Ser Gln Val Cys Glu Lys Val Thr Asn Ser Val Ser Ser Lys 
170                 175                 180                 185 

CTG CAA CCT TAT TTC CAG ACT CTG CCA GTA ATG ACC AAA ATA GAT TCT      726 
Leu Gln Pro Tyr Phe Gln Thr Leu Pro Val Met Thr Lys Ile Asp Ser 
                190                 195                 200 

GTG GCT GGA ATC AAC TAT GGT CTG GTG GCA CCT CCA GCA ACC ACG GCT      774 
Val Ala Gly Ile Asn Tyr Gly Leu Val Ala Pro Pro Ala Thr Thr Ala 
             205                 210                 215 

GAG ACC CTG GAT GTA CAG ATG AAG GGG GAG TTT TAC AGT GAG AAC CAC      822 
Glu Thr Leu Asp Val Gln Met Lys Gly Glu Phe Tyr Ser Glu Asn His 
        220                 225                 230 

CAC AAT CCA CCT CCC TTT GCT CCA CCA GTG ATG GAG TTT CCC GCT GCC      870 
His Asn Pro Pro Pro Phe Ala Pro Pro Val Met Glu Phe Pro Ala Ala 
    235                 240                 245 

CAT GAC CGC ATG GTA TAC CTG GGC CTC TCA GAC TAC TTC TTC AAC ACA      918 
His Asp Arg Met Val Tyr Leu Gly Leu Ser Asp Tyr Phe Phe Asn Thr 
250                 255                 260                 265 

GCC GGG CTT GTA TAC CAA GAG GCT GGG GTC TTG AAG ATG ACC CTT AGA      966 
Ala Gly Leu Val Tyr Gln Glu Ala Gly Val Leu Lys Met Thr Leu Arg 
                270                 275                 280 

GAT GAC ATG ATT CCA AAG GAG TCC AAA TTT CGA CTG ACA ACC AAG TTC     1014 
Asp Asp Met Ile Pro Lys Glu Ser Lys Phe Arg Leu Thr Thr Lys Phe 
            285                 290                 295 

TTT GGA ACC TTC CTA CCT GAG GTG GCC AAG AAG TTT CCC AAC ATG AAG     1062 
Phe Gly Thr Phe Leu Pro Glu Val Ala Lys Lys Phe Pro Asn Met Lys 
        300                 305                 310 

ATA CAG ATC CAT GTC TCA GCC TCC ACC CCG CCA CAC CTG TCT GTG CAG     1110 
Ile Gln Ile His Val Ser Ala Ser Thr Pro Pro His Leu Ser Val Gln 
     315                 320                 325 

CCC ACC GGC CTT ACC TTC TAC CCT GCC GTG GAT GTC CAG GCC TTT GCC     1158 
Pro Thr Gly Leu Thr Phe Tyr Pro Ala Val Asp Val Gln Ala Phe Ala 
330                 335                 340                 345 

GTC CTC CCC AAC TCC TCC CTG GCT TCC CTC TTC CTG ATT GGC ATG CAC     1206 
Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Phe Leu Ile Gly Met His 
                350                 355                 360 

ACA ACT GGT TCC ATG GAG GTC AGC GCC GAG TCC AAC AGG CTT GTT GGA     1254 
Thr Thr Gly Ser Met Glu Val Ser Ala Glu Ser Asn Arg Leu Val Gly 
            365                 370                 375 

GAG CTC AAG CTG GAT AGG CTG CTC CTG GAA CTG AAG CAC TCA AAT ATT     1302 
Glu Leu Lys Leu Asp Arg Leu Leu Leu Glu Leu Lys His Ser Asn Ile 
        380                 385                 390 

GGC CCC TTC CCG GTT GAA TTG CTG CAG GAT ATC ATG AAC TAC ATT GTA     1350 
Gly Pro Phe Pro Val Glu Leu Leu Gln Asp Ile Met Asn Tyr Ile Val 
    395                 400                 405 

CCC ATT CTT GTG CTG CCC AGG GTT AAC GAG AAA CTA CAG AAA GGC TTC     1398 
Pro Ile Leu Val Leu Pro Arg Val Asn Glu Lys Leu Gln Lys Gly Phe 
410                 415                 420                 425 

CCT CTC CCG ACG CCG GCC AGA GTC CAG CTC TAC AAC GTA GTG CTT CAG     1446 
Pro Leu Pro Thr Pro Ala Arg Val Gln Leu Tyr Asn Val Val Leu Gln 
                430                 435                 440 

CCT CAC CAG AAC TTC CTG CTG TTC GGT GCA GAC GTT GTC TAT AAA         1491 
Pro His Gln Asn Phe Leu Leu Phe Gly Ala Asp Val Val Tyr Lys 
            445                 450                 455 

TGAAGGCACC AGGGGTGCCG GGGGCTGTCA GCCGCACCTG TTCCTGATGG GCTGTGGG     1551 

ACCGGCTGCC TTTCCCCAGG GAATCCTCTC CAGATCTTAA CCAAGAGCCC CTTGCAAA     1611 

TCTTCGACTC AGATTCAGAA ATGATCTAAA CACGAGGAAA CATTATTCAT TGGAAAAG     1671 

CATGGTGTGT ATTTTAGGGA TTATGAGCTT CTTTCAAGGG CTAAGGCTGC AGAGATAT     1731 

CCTCCAGGAA TCGTGTTTCA ATTGTAACCA AGAAATTTCC ATTTGTGCTT CATGAAAA     1791 

AACTTCTGGT TTTTTTCATG TG                                            1813 

 
           
           
             
               487 amino acids  
               amino acid  
               linear  
             
             
               protein  
             
              265 

Met Arg Glu Asn Met Ala Arg Gly Pro Cys Asn Ala Pro Arg Trp Val 
-31 -30                 -25                 -20 

Ser Leu Met Val Leu Val Ala Ile Gly Thr Ala Val Thr Ala Ala Val 
-15                 -10                  -5                   1 

Asn Pro Gly Val Val Val Arg Ile Ser Gln Lys Gly Leu Asp Tyr Ala 
              5                  10                  15 

Ser Gln Gln Gly Thr Ala Ala Leu Gln Lys Glu Leu Lys Arg Ile Lys 
         20                  25                  30 

Ile Pro Asp Tyr Ser Asp Ser Phe Lys Ile Lys His Leu Gly Lys Gly 
     35                  40                  45 

His Tyr Ser Phe Tyr Ser Met Asp Ile Arg Glu Phe Gln Leu Pro Ser 
 50                  55                  60                  65 

Ser Gln Ile Ser Met Val Pro Asn Val Gly Leu Lys Phe Ser Ile Ser 
                 70                  75                  80 

Asn Ala Asn Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe 
             85                  90                  95 

Leu Lys Met Ser Gly Asn Phe Asp Leu Ser Ile Glu Gly Met Ser Ile 
         100                 105                 110 

Ser Ala Asp Leu Lys Leu Gly Ser Asn Pro Thr Ser Gly Lys Pro Thr 
    115                 120                 125 

Ile Thr Cys Ser Ser Cys Ser Ser His Ile Asn Ser Val His Val His 
130                 135                 140                 145 

Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys 
                150                 155                 160 

Ile Glu Ser Ala Leu Arg Asn Lys Met Asn Ser Gln Val Cys Glu Lys 
            165                 170                 175 

Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln Thr Leu 
        180                 185                 190 

Pro Val Met Thr Lys Ile Asp Ser Val Ala Gly Ile Asn Tyr Gly Leu 
    195                 200                 205 

Val Ala Pro Pro Ala Thr Thr Ala Glu Thr Leu Asp Val Gln Met Lys 
210                 215                 220                 225 

Gly Glu Phe Tyr Ser Glu Asn His His Asn Pro Pro Pro Phe Ala Pro 
                230                 235                 240 

Pro Val Met Glu Phe Pro Ala Ala His Asp Arg Met Val Tyr Leu Gly 
            245                 250                 255 

Leu Ser Asp Tyr Phe Phe Asn Thr Ala Gly Leu Val Tyr Gln Glu Ala 
        260                 265                 270 

Gly Val Leu Lys Met Thr Leu Arg Asp Asp Met Ile Pro Lys Glu  Ser 
    275                 280                 285 

Lys Phe Arg Leu Thr Thr Lys Phe Phe Gly Thr Phe Leu Pro Glu Val 
290                 295                 300                 305 

Ala Lys Lys Phe Pro Asn Met Lys Ile Gln Ile His Val Ser Ala Ser 
                310                 315                 320 

Thr Pro Pro His Leu Ser Val Gln Pro Thr Gly Leu Thr Phe Tyr Pro 
            325                 330                 335 

Ala Val Asp Val Gln Ala Phe Ala Val Leu Pro Asn Ser Ser Leu Ala 
        340                 345                 350 

Ser Leu Phe Leu Ile Gly Met His Thr Thr Gly Ser Met Glu Val Ser 
    355                 360                 365 

Ala Glu Ser Asn Arg Leu Val Gly Glu Leu Lys Leu Asp Arg Leu Leu 
370                 375                 380                 385 

Leu Glu Leu Lys His Ser Asn Ile Gly Pro Phe Pro Val Glu Leu Leu 
                390                 395                 400 

Gln Asp Ile Met Asn Tyr Ile Val Pro Ile Leu Val Leu Pro Arg Val 
            405                 410                 415 

Asn Glu Lys Leu Gln Lys Gly Phe Pro Leu Pro Thr Pro Ala Arg Val 
        420                 425                 430 

Gln Leu Tyr Asn Val Val Leu Gln Pro His Gln Asn Phe Leu Leu Phe 
    435                 440                 445 

Gly Ala Asp Val Val Tyr Lys 
450                 455