Source: https://patents.google.com/patent/US7732569
Timestamp: 2018-03-22 06:26:52
Document Index: 778998421

Matched Legal Cases: ['§119', '§1', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60', 'Application No. 60']

US7732569B2 - Zein-based peptide tags for the expression and purification of bioactive peptides - Google Patents
Zein-based peptide tags for the expression and purification of bioactive peptides Download PDF
US7732569B2
US7732569B2 US11641936 US64193606A US7732569B2 US 7732569 B2 US7732569 B2 US 7732569B2 US 11641936 US11641936 US 11641936 US 64193606 A US64193606 A US 64193606A US 7732569 B2 US7732569 B2 US 7732569B2
US11641936
US20080096246A1 (en )
Zein-based peptide tags, referred to here as inclusion body tags (IBTs), are disclosed useful for the generation of insoluble fusion peptides. The fusion peptides comprise at least one inclusion body tag operably linked to a peptide of interest. Expression of the fusion peptide in a host cell results in a product that is insoluble and contained within inclusion bodies in the cell and/or cell lysate. The inclusion bodies may then be purified and the protein of interest may be isolated after cleavage from the inclusion body tag.
This application claims priority under 35 U.S.C.§119 from U.S. Provisional Application Ser. No. 60/852,838, filed Oct. 19, 2006.
Fusion proteins comprising a carrier protein tag that facilitates the expression of insoluble proteins are well known in the art. Typically, the tag portion of the chimeric or fusion protein is large, increasing the likelihood that the fusion protein will be insoluble. Example of large peptide tags typically used include, but are not limited to chloramphenicol acetyltransferase (Dykes et al., Eur. J. Biochem., 174:411 (1988), β-galactosidase (Schellenberger et al., Int. J. Peptide Protein Res., 41:326 (1993); Shen et al., Proc. Nat. Acad. Sci. USA 281:4627 (1984); and Kempe et al., Gene, 39:239 (1985)), glutathione-S-transferase (Ray et al., Bio/Technology, 11:64 (1993) and Hancock et al. (WO94/04688)), the N-terminus of L-ribulokinase (U.S. Pat. No. 5,206,154 and Lai et al., Antimicrob. Agents & Chemo., 37:1614 (1993), bacteriophage T4 gp55 protein (Gramm et al., Bio/Technology, 12:1017 (1994), bacterial ketosteroid isomerase protein (Kuliopulos et al., J. Am. Chem. Soc. 116:4599 (1994), ubiquitin (Pilon et al., Biotechnol. Prog., 13:374-79 (1997), bovine prochymosin (Haught et al., Biotechnol. Bioengineer. 57:55-61 (1998), and bactericidal/permeability-increasing protein (“BPI”; Better, M. D. and Gavit, P D., U.S. Pat. No. 6,242,219). The art is replete with specific examples of this technology, see for example U.S. Pat. No. 6,613,548, describing fusion protein of proteinaceous tag and a soluble protein and subsequent purification from cell lysate; U.S. Pat. No. 6,037,145, teaching a tag that protects the expressed chimeric protein from a specific protease; U.S. Pat. No. 5,648,244, teaching the synthesis of a fusion protein having a tag and a cleavable linker for facile purification of the desired protein; and U.S. Pat. No. 5,215,896; 5,302,526; 5,330,902; and US 2005221444, describing fusion tags containing amino acid compositions specifically designed to increase insolubility of the chimeric protein or peptide.
The stated problem has been solved though the discovery of a set of short inclusion body tags (IBTs) derived from a Zea mays zein protein that are useful for synthesizing fusion proteins for increased expression and simple purification of short peptides (“peptides of interest”), especially short peptides useful in affinity applications.
Accordingly, the present invention provides an inclusion body tag comprising at least 15 contiguous amino acids from residues 76 to 175 of SEQ ID NO: 2 with the proviso that the inclusion body tag is not SEQ ID NO: 2.
In another aspect, the invention provides a fusion peptide comprising the inclusion body tag of the invention operably linked to a peptide of interest. The inclusion body tag can be a leader or trailer sequence within the fusion protein. In a preferred aspect, the fusion peptide is engineered to include at least one cleavable peptide linker. Inclusion of a cleavable peptide linker is useful for separating the inclusion body tag and the peptide of interest. In another preferred aspect, the cleavable peptide linker comprises at least one acid cleavable aspartic acid—proline (DP) moiety.
The following sequences comply with 37 C.F.R. 1.821-1.825 (“Requirements for Patent Applications Containing Nucleotide Sequences and/or Amino Acid Sequence Disclosures—the Sequence Rules”) and are consistent with World Intellectual Property Organization (WIPO) Standard ST.25 (1998) and the sequence listing requirements of the EPC and PCT (Rules 5.2 and 49.5(a-bis), and Section 208 and Annex C of the Administrative Instructions). The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R.§1.822.
A Sequence Listing is provided herewith on Compact Disk. The contents of the Compact Disk containing the Sequence Listing are hereby incorporated by reference in compliance with 37 CFR 1.52(e). The Compact Disks are submitted in triplicate and are identical to one another. The disks are labeled “Copy 1—Sequence Listing”, “Copy 2—Sequence Listing”, and CRF. The disks contain the following file: CL3262 US NA.ST25 having the following size: 209,000 bytes and which was created Nov. 30, 2006.
SEQ ID NO: 1 is the nucleotide sequence of the opaque2 modifier (also referred to herein as “gamma zeinA”) coding region from Zea mays.
SEQ ID NO: 2 is the amino acid sequence of the 27 kDa gamma zeinA protein (GenBank® AAP32017).
SEQ ID NO: 12 is the nucleotide sequence of pENTR™/D-TOPO® plasmid (Invitrogen, Carlsbad, Calif.).
SEQ ID NOs: 21-110 are the nucleotide sequences of oligonucleotides used to prepare the present inclusion body tags.
SEQ ID NOs: 111-155 are the amino acid sequences of peptides evaluated as potential inclusion body tags.
SEQ ID NOs: 156-245 are the nucleotide and corresponding amino acid sequences of the fusion proteins created by fusing the present inclusion body tags to the modified TBP101 peptide.
SEQ ID NO: 246 is the amino acid sequence of the T7 translational enhancer.
SEQ ID NO: 247 is the amino acid sequence of inclusion body tag IBT-180.
SEQ ID NO: 248 is the amino acid sequence of inclusion body tag IBT-181.
SEQ ID NO: 249 is the nucleic acid sequence of the chimeric gene IBT 180-TBP101.
SEQ ID NO: 250 is the amino acid sequence of the fusion peptide IBT 180-TBP101.
SEQ ID NO: 251 is the nucleic acid sequence of the chimeric gene IBT 181-TBP101.
SEQ ID NO: 252 is the amino acid sequence of the fusion peptide IBT 181-TBP101.
SEQ ID NOs: 253-355 are examples of amino acid sequences of body surface binding peptides, SEQ ID NOs 253-260 are skin binding peptides, SEQ ID NOs 261-353 are hair binding peptides, and SEQ ID NOs: 354-355 are nail binding peptides.
SEQ ID NOs: 356-384 are examples of antimicrobial peptide sequences.
SEQ ID NOs: 385-410 are examples of pigment binding peptides, SEQ ID NOs: 385-388 bind carbon black, SEQ ID NOs: 389-397 are Cromophtal® yellow (Ciba Specialty Chemicals, Basel, Switzerland) binding peptides, SEQ ID NOs: 398-400 are Sunfast® magenta (Sun Chemical Corp., Parsippany, N.J.) binding peptides, and SEQ ID NOs: 401-410 are Sunfast® blue binding peptides.
SEQ ID NOs: 411-444 are examples of polymer binding peptides, SEQ ID NOs: 411-416 are cellulose binding peptides, SEQ ID NO: 417 is a poly(ethylene terephthalate) (PET) binding peptide, SEQ ID NOs: 418-429 are poly(methyl methacrylate) (PMMA) binding peptides, SEQ ID NOs: 430-435 are nylon binding peptides, and SEQ ID NOs: 436-444 are poly(tetrafluoro ethylene) (PTFE) binding peptides.
SEQ ID NO: 445 is the amino acid sequence of the Caspase-3 cleavage site that may be used as a cleavable peptide linker domain.
As used herein, the term “nails” as used herein refers to human fingernails and toenails and other body surfaces comprised primarily of keratin.
As used herein, “HBP” means hair-binding peptide. Examples of hair binding peptides have been reported (U.S. patent application Ser. No. 11/074,473 to Huang et al.; WO 0179479; U.S. Patent Application Publication No. 2002/0098524 to Murray et al.; Janssen et al., U.S. Patent Application Publication No. 2003/0152976 to Janssen et al.; WO 04048399; U.S. Provisional Application No. 60/721,329, and U.S. Provisional Patent Application No. 60/790,149).
As used herein, the terms “zein 27 kDa storage protein”, “zein protein”, “gamma zein protein”, and “opaque2 protein” will refer to the Zea mays protein having the amino acid sequence as set forth in SEQ ID NO:2 (GenBank® Accession No. AAP32017). The coding region encoding the zein protein having GenBank® Accession No. AAP32017 is provided as SEQ ID NO: 1.
As used herein, the term “inclusion body tag” will be abbreviated “IBT” and will refer a polypeptide that facilitates/stimulates formation of inclusion bodies when fused to a peptide of interest. The peptide of interest is typically soluble within the host cell and/or host cell lysate when not fused to an inclusion body tag. Fusion of the peptide of interest to the inclusion body tag produces an insoluble fusion protein that typically agglomerates into intracellular bodies (inclusion bodies) within the host cell. In one embodiment, the fusion protein comprises at least one portion comprising an inclusion body tag and at least one portion comprising the polypeptide of interest. In one embodiment, the protein/polypeptides of interest are separated from the inclusion body tags using cleavable peptide linker elements.
As used herein, “T7 translational enhancer element” means the N-terminal coding sequence of bacteriophage T7 gene 10 (Rosenberg, A H et al., Gene 56:125-135 (1987)), which provides a standardized sequence at the critical translation initiation site in the genes encoding IBT-180 and IBT-181.
As used herein, “cleavable linker elements”, “peptide linkers”, and “cleavable peptide linkers” will be used interchangeably and refer to cleavable peptide segments typically found between inclusion body tags and the peptide of interest. After the inclusion bodies are separated and/or partially-purified or purified from the cell lysate, the cleavable linker elements can be cleaved chemically and/or enzymatically to separate the inclusion body tag from the peptide of interest. The peptide of interest can then be isolated from the inclusion body tag, if necessary. In one embodiment, the inclusion body tag(s) and the peptide of interest exhibit different solubilities in a defined medium (typically an aqueous medium), facilitating separation of the inclusion body tag from the protein/polypeptide of interest. In a preferred embodiment, the inclusion body tag is insoluble in an aqueous solution while the protein/polypeptide of interest is appreciably soluble in an aqueous solution. The pH, temperature, and/or ionic strength of the aqueous solution can be adjusted to facilitate recovery of the peptide of interest. In a preferred embodiment, the differential solubility between the inclusion body tag and the peptide of interest occurs in an aqueous solution having a pH of 5 to 10 and a temperature range of 15 to 50° C. The cleavable peptide linker may be from 1 to about 50 amino acids, preferably from 1 to about 20 amino acids in length. An example of a cleavable peptide linker is provided by SEQ ID NO: 445 (Caspase-3 cleavage sequence). The cleavable peptide linkers may be incorporated into the fusion proteins using any number of techniques well known in the art.
As used herein, the term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). In a further embodiment, the definition of “operably linked” may also be extended to describe the products of chimeric genes, such as fusion proteins. As such, “operably linked” will also refer to the linking of an inclusion body tag to a peptide of interest to be produced and recovered. The inclusion body tag is “operably linked” to the peptide of interest if upon expression the fusion protein is insoluble and accumulates it inclusion bodies in the expressing host cell. In a preferred embodiment, the fusion peptide will include at least one cleavable peptide linker useful in separating the inclusion body tag from the peptide of interest. In a further preferred embodiment, the cleavable linker is an acid cleavable aspartic acid—proline dipeptide (D-P) moiety (see INK101DP; SEQ ID NO: 20). The cleavable peptide linkers may be incorporated into the fusion proteins using any number of techniques well known in the art.
As used herein, the terms “fusion protein”, “fusion peptide”, “chimeric protein”, and “chimeric peptide” will be used interchangeably and will refer to a polymer of amino acids (peptide, oligopeptide, polypeptide, or protein) comprising at least two portions, each portion comprising a distinct function. One portion of the fusion peptide will comprise at least one of the present inclusion body tags. The second portion comprises at least one peptide of interest. In a preferred embodiment, the fusion protein additionally includes at least one cleavable peptide linker that facilitates cleavage (chemical and/or enzymatic) and separation of the inclusion body tag(s) and the peptide(s) of interest.
Means to prepare the present peptides (inclusion body tags, cleavable peptide linkers, peptides of interest, and fusion peptides) are well known in the art (see, for example, Stewart et al., Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, Ill., 1984; Bodanszky, Principles of Peptide Synthesis, Springer-Verlag, N.Y., 1984; and Pennington et al., Peptide Synthesis Protocols, Humana Press, Totowa, N.J., 1994). The various components of the fusion peptides (inclusion body tag, peptide of interest, and the cleavable linker) described herein can be combined using carbodiimide coupling agents (see for example, Hermanson, Greg T., Bioconjugate Techniques, Academic Press, New York (1996)), diacid chlorides, diisocyanates and other difunctional coupling reagents that are reactive to terminal amine and/or carboxylic acid groups on the peptides. However, chemical synthesis is often limited to peptides of less than about 50 amino acids length due to cost and/or impurities. In a preferred alternative embodiment, the entire peptide reagent may be prepared using the recombinant DNA and molecular cloning techniques.
As used herein, the terms “bioactive” and “peptide of interest activity” are used interchangeably and refer to the activity or characteristic associated with the peptide and/or protein of interest. The bioactive peptides may be used in a variety of applications including, but not limited to curative agents for diseases (e.g., insulin, interferon, interleukins, anti-angiogenic peptides (U.S. Pat. No. 6,815,426), and polypeptides that bind to defined cellular targets such as receptors, channels, lipids, cytosolic proteins, and membrane proteins, to name a few), peptides having antimicrobial activity, peptides having an affinity for a particular material (e.g., hair binding polypeptides, skin binding polypeptides, nail binding polypeptides, cellulose binding polypeptides, polymer binding polypeptides, clay binding polypeptides, silicon binding polypeptides, carbon nanotube binding polypeptides, and peptides that have an affinity for particular animal or plant tissues) for targeted delivery of benefit agents.
As used herein, the term “solubility” refers to the amount of a substance that can be dissolved in a unit volume of a liquid under specified conditions. In the present application, the term “solubility” is used to describe the ability of a peptide (inclusion body tag, peptide of interest, or fusion peptides) to be resuspended in a volume of solvent, such as a biological buffer. In one embodiment, the peptides targeted for production (“peptides of interest”) are normally soluble in the cell and/or cell lysate under normal physiological conditions. Fusion of one or more inclusion body tags (IBTs) to the target peptide results in the formation of a fusion peptide that is insoluble under normal physiological conditions, resulting in the formation of inclusion bodies. In one embodiment, the peptide of interest is insoluble in an aqueous matrix having a pH range of 5-12, preferably 6-10; and a temperature range of 5° C. to 5° C., preferably 10° C. to 40° C. Fusion of the peptide of interest to at least one of the present inclusion body tags results in the formation of an insoluble fusion protein that agglomerates into at least one inclusion body under normal physiological conditions.
Miscellaneous (as Xaa X
As used herein, the term “coding sequence” refers to a DNA sequence that encodes for a specific amino acid sequence. “Suitable regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, enhancers, ribosomal binding sites, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing site, effector binding site and stem-loop structures. One of skill in the art recognizes that selection of suitable regulatory sequences will depend upon host cell and/or expression system used.
As used herein, the term “transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. As used herein, the host cell's genome is comprised of chromosomal and extrachromosomal (e.g., plasmid) genes. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” or “recombinant” or “transformed” organisms.
The inclusion body tags of the invention were derived from the gamma zein 27kDA storage protein (GenBank® accession No. AAP32017; SEQ ID NO: 2). This protein was selected as the starting material for preparation of a library of putative inclusion body tags. Several overlapping series of approximately 15 amino acid long peptides (one was 13 amino acids in length) were prepared and evaluated as potential inclusion body tags. The library was prepared by synthesizing and fusing short peptides identical to various portions of the zein protein to a soluble peptide of interest (the modified TBP101 peptide). Expression analysis identified a central region of the zein protein (amino acid residues 76 through 175 of SEQ ID NO: 2) suitable for the preparation of the present inclusion body tags. Short (15 or more contiguous amino acids) inclusion body tags prepared from this region were able to induce inclusion body formation (i.e. form insoluble fusion peptides) when fused to a peptide of interest (typically soluble).
Each of the present fusion tags was fused to a standard peptide of interest (a modified version of TBP101 incorporating an acid cleavable aspartic acid—proline moiety useful in separating the peptide of interest from the inclusion body tag; see Example 1). TBP101 (when not linked to an inclusion body tag) is soluble in the present test system. Each constructed was recombinantly expressed in an appropriate host cell and evaluated for insoluble fusion peptide formation.
The present inclusion body tags are peptides comprising at least 15 contiguous amino acid from amino acid residues 76 to 175 of SEQ ID NO: 2 with the proviso that the inclusion body tag is not equal to SEQ ID NO: 2 or any other full length zein protein. In one embodiment, the inclusion body tags may comprise additional amino acid residues flanking the present peptide sequences so long as the ability to form insoluble fusion peptides in not adversely affected with the proviso that the amino acid sequence of the inclusion body tag is not identical to SEQ ID NO: 2. In another embodiment, portion of the fusion protein comprising the inclusion body tag of the present invention is 15 to no more than 100 amino acid residues in length, preferably 15 to 50 amino acids in length, more preferably 15 to 25 amino acids in length, and most preferably about 15 amino acids in length.
In one embodiment, the present inclusion body tags are selected from the group consisting of SEQ ID NOs: 116, 117, 119, 121, 131, 132, 133, 135, 145, 147, 148, 149, 150, 154, 155, 247, and 248.
Inclusion body tags IBT-180 and IBT-181 each include a T7 translational enhancer element (SEQ ID NO: 246) fused to the amino terminal portion of an inclusion body tag derived the zein protein. In one aspect, any of the present inclusion body tags may optionally include the T7 translational enhancer as represented by SEQ ID NO: 246 fused to the amino terminus of an inclusion body tag comprising at least 15 contiguous amino acids from amino acid residues 76 to 175 of SEQ ID NO: 2.
In another aspect, the present invention also includes fusion peptides comprising at least one of the present inclusion body tags fused to at least one peptide of interest. In a preferred embodiment, the fusion peptide includes at least one cleavable peptide linker useful in separating the peptide of interest from the inclusion body tag(s). The cleavable peptide linker can be an enzymatic cleavage sequence or a chemically cleavable sequence. In another preferred embodiment, the cleavable peptide linker comprises at least one acid cleavable aspartic acid—proline moiety (for example, see the INK101DP peptide; SEQ ID NO: 20).
In a preferred aspect, the peptide of interest is selected from the group of hair binding peptides (U.S. patent application Ser. No. 11/074,473; WO 0179479; U.S. Patent Application Publication No. 2002/0098524; Janssen et al., U.S. Patent Application Publication No. 2003/0152976; WO 04048399; U.S. Provisional Patent Application No. 60/721,329; and U.S. Provisional Patent Application No. 60/790,149), skin binding peptides (U.S. patent application Ser. No. 11/069,858; WO 2004/000257; and U.S. Provisional Patent Application No. 60/790,149), nail binding peptides (U.S. Provisional Patent Application No. 60/790,149), antimicrobial peptides (U.S. Provisional Patent Application No. 60/790,149), and polymer binding peptides (U.S. Provision Patent Application Nos. 60/750,598, 60/750,599, 60/750,726, 60/750,748, and 60/750,850). In another preferred aspect, the hair binding peptide is selected from the group consisting of SEQ ID NOs: (261-353); the skin binding peptide is selected from the group consisting of SEQ ID NOs: (253-260); the nail binding peptide is selected from the group consisting of SEQ ID NOs: (354-355); the antimicrobial peptide is selected from the group consisting of SEQ ID NOs: (356-384); and the polymer binding peptide is selected from the group consisting of SEQ ID NOs: (411-444).
As used herein, the “benefit agent” refers to a molecule that imparts a desired functionality to a target material (e.g., hair, skin, etc.) for a defined application (U.S. patent application Ser. No. 10/935,642; U.S. patent application Ser. No. 11/074,473; and U.S. Patent Application 60/790,149 for a list of typical benefit agents such as conditioners, pigments/colorants, fragrances, etc.). The benefit agent may be the peptide of interest itself or may be one or more molecules bound to (covalently or non-covalently), or associated with, the peptide of interest wherein the binding affinity of the peptide of interest is used to selectively target the benefit agent to the targeted material. In another embodiment, the peptide of interest comprises at least one region having an affinity for at least one target material (e.g., biological molecules, polymers, hair, skin, nail, other peptides, etc.) and at least one region having an affinity for the benefit agent (e.g., pharmaceutical agents, antimicrobial agents, pigments, conditioners, dyes, fragrances, etc.). In another embodiment, the peptide of interest comprises a plurality of regions having an affinity for the target material and a plurality of regions having an affinity for one or more benefit agents. In yet another embodiment, the peptide of interest comprises at least one region having an affinity for a targeted material and a plurality of regions having an affinity for a variety of benefit agents wherein the benefit agents may be the same of different. Examples of benefits agents may include, but are not limited to conditioners for personal care products, pigments, dye, fragrances, pharmaceutical agents (e.g., targeted delivery of cancer treatment agents), diagnostic/labeling agents, ultraviolet light blocking agents (i.e., active agents in sunscreen protectants), and antimicrobial agents (e.g., antimicrobial peptides), to name a few.
The use of cleavable peptide linkers is well known in the art. Fusion peptides comprising the present inclusion body tags will typically include at least one cleavable sequence separating the inclusion body tag from the polypeptide of interest. The cleavable sequence facilitates separation of the inclusion body tag(s) from the peptide(s) of interest. In one embodiment, the cleavable sequence may be provided by a portion of the inclusion body tag and/or the peptide of interest (e.g., inclusion of an acid cleavable aspartic acid—proline moiety; see INK101 DP (SEQ ID NO: 20)). In a preferred embodiment, the cleavable sequence is provided by including (in the fusion peptide) at least one cleavable peptide linker between the inclusion body tag and the peptide of interest.
In another embodiment, one or more enzymatic cleavage sequences are included in the fusion protein construct to facilitate recovery of the peptide of interest. Proteolytic enzymes and their respective cleavage site specificities are well known in the art. In a preferred embodiment, the proteolytic enzyme is selected to specifically cleave only the peptide linker separating the inclusion body tag and the peptide of interest. Examples of enzymes useful for cleaving the peptide linker include, but are not limited to Arg-C proteinase, Asp-N endopeptidase, chymotrypsin, clostripain, enterokinase, Factor Xa, glutamyl endopeptidase, Granzyme B, Achromobacter proteinase I, pepsin, proline endopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin, thrombin, trypsin, and members of the Caspase family of proteolytic enzymes (e.g. Caspases 1-10) (Walker, J. M., supra). An example of a cleavage site sequence is provided by SEQ ID NO: 445 (Caspase-3 cleavage site; Thornberry et al., J. Biol. Chem., 272:17907-17911 (1997) and Tyas et al., EMBO Reports, 1 (3):266-270 (2000)).
Initiation control regions or promoters, which are useful to drive expression of the genetic constructs encoding the fusion peptides in the desired host cell, are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these constructs is suitable for the present invention including but not limited to CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, ara (pBAD), tet, trp, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus.
Preferred host cells for expression of the present fusion peptides are microbial hosts that can be found broadly within the fungal or bacterial families and which grow over a wide range of temperature, pH values, and solvent tolerances. For example, it is contemplated that any of bacteria, yeast, and filamentous fungi will be suitable hosts for expression of the present nucleic acid molecules encoding the fusion peptides. Because of transcription, translation, and the protein biosynthetic apparatus is the same irrespective of the cellular feedstock, genes are expressed irrespective of the carbon feedstock used to generate the cellular biomass. Large-scale microbial growth and functional gene expression may utilize a wide range of simple or complex carbohydrates, organic acids and alcohols (i.e. methanol), saturated hydrocarbons such as methane or carbon dioxide in the case of photosynthetic or chemoautotrophic hosts. However, the functional genes may be regulated, repressed or depressed by specific growth conditions, which may include the form and amount of nitrogen, phosphorous, sulfur, oxygen, carbon or any trace micronutrient including small inorganic ions. In addition, the regulation of functional genes may be achieved by the presence or absence of specific regulatory molecules that are added to the culture and are not typically considered nutrient or energy sources. Growth rate may also be an important regulatory factor in gene expression. Examples of host strains include, but are not limited to fungal or yeast species such as Aspergillus, Trichoderma, Saccharomyces, Pichia, Candida, Hansenula, or bacterial species such as Salmonella, Bacillus, Acinetobacter, Zymomonas, Agrobacterium, Erythrobacter, Chlorobium, Chromatium, Flavobacterium, Cytophaga, Rhodobacter, Rhodococcus, Streptomyces, Brevibacterium, Corynebacteria, Mycobacterium, Deinococcus, Escherichia, Erwinia, Pantoea, Pseudomonas, Sphingomonas, Methylomonas, Methylobacter, Methylococcus, Methylosinus, Methylomicrobium, Methylocystis, Alcaligenes, Synechocystis, Synechococcus, Anabaena, Thiobacillus, Methanobacterium, Klebsiella, and Myxococcus. Preferred bacterial host strains include Escherichia and Bacillus. In a highly preferred aspect, the host strain is Escherichia coli.
The meaning of abbreviations used is as follows: “min” means minute(s), “h” means hour(s), “μL” means microliter(s), “mL” means milliliter(s), “L” means liter(s), “nm” means nanometer(s), “mm” means millimeter(s), “cm” means centimeter(s), “μm” means micrometer(s), “mM” means millimolar, “M” means molar, “mmol” means millimole(s), “μmole” means micromole(s), “pmol” means picomole(s), “g” means gram(s), “μg” means microgram(s), “mg” means milligram(s), “g” means the gravitation constant, “rpm” means revolutions per minute, “DTT” means dithiothreitol, and “cat#” means catalog number.
Example 1 Preparation of Plasmid pLX121 for Evaluating Inclusion Body Tag Performance
Name Nucleotide Sequence (5′-3′) NO:
TBP1(+)1 GGATCCATCGAAGGTCGTTTCCACGAA 5
TBP1(+)2 CCTCTAAGACTACCACGACTACCTCCAA 6
TBP1(−)1 TTATGCAGCCAGCAGACGCTGAGTAGAG 7
TBP1(−)2 AGAGGTTTTGGAGGTAGTCGTGGTAGTC 8
TBP1(−)3 GGAAGCTTTGGAAGTAGAGGTACCGC 9
GAGCTTCGATGGATCC
Lambda phage site-specific recombination was used for preparation and expression of the present fusion proteins (Gateway™ System; Invitrogen, Carlsbad, Calif.). TBP1 was integrated into the Gateway™ system for protein over-expression. In the first step, 2 μL of the TBP1 ligation mixture was used in a 50-μL PCR reaction. Reactions were catalyzed by pfu DNA polymerase (Stratagene, La Jolla, Calif.), following the standard PCR protocol. Primer 5′TBP1 (5′-CACCGGATCCATCGAAGGTCGT-3′; SEQ ID NO: 10) and 3′TBP1 (5′-TCATTATGCAGCCAGCAGCGC-3′; SEQ ID NO: 11) were used for amplification of the TBP1 fragment. Due to the design of these primers, an additional sequence of CACC and another stop codon TGA were added to the 5′ and 3′ ends of the amplified fragments.
The amplified TBP1 was directly cloned into pENTR™/D-TOPO® vector (SEQ ID NO: 12) using Invitrogen's pENTR™ directional TOPO® cloning kit (Invitrogen; Catalog K2400-20), resulting in the Gateway™ entry plasmid pENTR-TBP1. This entry plasmid was propagated in One Shot® TOP10 E. coli cells (Invitrogen). The accuracy of the PCR amplification and cloning procedures were confirmed by DNA sequencing analysis. The entry plasmid was mixed with pDEST17 (Invitrogen, SEQ ID NO: 13). LR recombination reactions were catalyzed by LR Clonase™ (Invitrogen). The destination plasmid, pINK101 was constructed and propagated in the DH5α E. coli strain. The accuracy of the recombination reaction was determined by DNA sequencing. All reagents for LR recombination reactions (i.e., lambda phage site-specific recombination) were provided in Invitrogen's E. coli expression system with the Gateway™ Technology kit. The site-specific recombination process followed the manufacturer's instructions (Invitrogen).
The resulting plasmid, named pINK101, contains the coding regions for recombinant protein 6H-TBP1, named INK101 (SEQ ID NOs 14 and 15), which is an 11.6 kDa protein. The protein sequence includes a 6× His tag and a 24 amino acid linker that includes Factor Xa protease recognition site before the sequence of the TBP101 peptide.
The amino acid coding region for the 6× His tag and the following linker comprising the Factor Xa protease recognition site were excised from pINK101 by digestion with the NdeI and BamHI restriction enzymes.
Further modifications were made to TBP101 including the addition of an acid cleavable site to facilitate the removal of any tag sequence encoded by the region between the NdeI and BamHI sites of the expression plasmid. The resulting plasmid was called pLX121 (also referred to as “pINK101 DP”; SEQ ID NO: 16). These modifications changed the amino acids E-G to D-P (acid cleavable aspartic acid—proline linkage) using the Stratagene QuikChange® II Site-Directed Mutagenesis Kit Cat# 200523 (La Jolla, Calif.) as per the manufacturer's protocol using the primers INK101+ (5′-CCCCTTCACCGGATCCATCGATCCACGTTTCCACGAAAACTGGCC-3′; SEQ ID 17) and INK101− (5′-GGCCAGTTTTCGTGGAAACGTGGATCGATGGATCCGGTGMGGGG-3′; SEQ ID NO 18). The sequences were confirmed by DNA sequence analysis. The coding region and the corresponding amino acid sequence of the modified protein, INK101DP, is provided as SEQ ID NOs 19 and 20, respectively. INK101DP (also referred to herein as “TBP101 DP”) was used to evaluate the present inclusion body tags.
INK101DP Peptide (SEQ ID NO: 20)
MSYYHHHHHHLESTSLYKKAGSAAAPFTGSI DP RFHENWPSAGGTSTS
KASTTTTSSKTTTTSSKTTTTTSKTSTTSSSSTGGATHKTSTQRLLAA
The aspartic acid—proline acid cleavable linker is bolded. The DP moiety replaces the EG moiety found in the unmodified TBP101 peptide (SEQ ID, NO: 4). The modified TBP101 peptide is underlined.
Example 2 Generation of Zein-based Inclusion Body Tag Library
Several series of inclusion body tag libraries were generated from the Zea mays zein storage protein (GenBank® Accession No. AAP32017; SEQ ID NO: 2 encoded by the coding sequence as represented by SEQ ID NO:1). Three series of putative inclusion body tags (typically 15 amino acids in length; one being only 13 amino acids in length) were prepared from 15 amino acid segments of the zein protein. Library series #1 (IBTs 65-79) was prepared by creating a set of 15 amino acid long peptides (IBT-79 is only 13 amino acids in length) from consecutive sequences spanning the entire length of the zein protein starting with amino acid residue position 1 of SEQ ID NO: 2 (i.e. IBT−65=amino acid residues 1-15 of SEQ ID NO: 2, IBT−66=amino acid residues 16-30 of SEQ ID NO: 2, etc.). Library series #2 (IBTs 80-121) was prepared in a similar fashion, except that the first member of the library series started with amino acid residue position 6 of SEQ ID NO: 2. Library series #3 (IBTs 122-135) was also prepared in a similar fashion starting at amino acid position 11 of SEQ ID NO: 2. In this way, an overlapping library 13-15 amino acid long peptides were prepared that spanned the entire length of zein protein (Table 2).
Based on the expression ranking data (i.e. the ability of the inclusion body tag to induce insoluble fusion protein when fused to a normally soluble peptide of interest; see Example 3), several additional inclusion body tags (IBTs 158-159) of varying length were prepared from regions of the zein protein suitable for use as inclusion body tags (Table 2).
Synthesis and Cloning Procedure for Preparing Inclusion Body Tags
The inclusion body tags were assembled from two complementary synthetic E. coli biased oligonucleotides (Sigma Genosys). Overhangs were included in each oligonucleotide to generate cohesive ends compatible with the restriction sites NdeI and BamHI.
The oligonucleotides (Table 2) were annealed by combining 100 pmol of each oligonucleotide in deionized water into one tube and heated in a water bath set at 99° C. for 10 minutes after which the water bath was turned off. The oligonucleotides were allowed to anneal slowly until the water bath reached room temperature (20-25° C.). The annealed oligonucleotides were diluted in 100 μL water prior to ligation into the test vector. The vector pLX121 (SEQ ID NO: 16) comprises the open reading frame encoding the INK101DP peptide (SEQ ID NO: 20). The vector was digested in Buffer 2 (New England Biolabs, Beverly, Mass.) comprising 10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol (DTT); pH ˜7.9) with the NdeI and BamHI restriction enzymes to release a 90 bp fragment corresponding to the original His6 containing inclusion body fusion partner and the linker from the parental pDEST17 plasmid that includes the att site of the Gateway™ Cloning System. The NdeI-BamHI fragments from the digested plasmid were separated by agarose gel electrophoresis and the vector was purified from the gel by using Qiagen QIAquick® Gel Extraction Kit (QIAGEN Valencia, Calif.; cat# 28704).
The diluted and annealed oligonucleotides (approximately 0.2 pmol) were ligated with T4 DNA Ligase (New England Biolabs Beverly, Mass.; catalog # M0202) to NdeI-BamHI digested, gel purified, plasmid pLX121 (approximately 50 ng) at 12° C. for 18 hours. DNA sequence analysis confirmed the expected plasmid sequence.
IBT Amino Amino Acid
Sequence Positions of the
Inclusion DNA Oligonucleotide (SEQ Zein Protein
Body Tag strand (SEQ ID NO.) ID NO.) (SEQ ID NO: 2)
IBT-65 + 21 111 1-15
IBT-65 − 22
IBT-66 + 23 112 16-30
IBT-66 − 24
IBT-67 + 25 113 31-45
IBT-67 − 26
IBT-68 + 27 114 46-60
IBT-68 − 28
IBT-69 + 29 115 61-75
IBT-69 − 30
IBT-70 + 31 116 76-90
IBT-70 − 32
IBT-71 + 33 117 91-105
IBT-71 − 34
IBT-72 + 35 118 106-120
IBT-72 − 36
IBT-73 + 37 119 121-135
IBT-73 − 38
IBT-74 + 39 120 136-150
IBT-74 − 40
IBT-75 + 41 121 151-165
IBT-75 − 42
IBT-76 + 43 122 166-180
IBT-76 − 44
IBT-77 + 45 123 181-195
IBT-77 − 46
IBT-78 + 47 124 196-210
IBT-78 − 48
IBT-79 + 49 125a 211-223a
IBT-79 − 50
IBT-108 + 51 126 6-20
IBT-108 − 52
IBT-109 + 53 127 21-35
IBT-109 − 54
IBT-110 + 55 128 36-50
IBT-110 − 56
IBT-111 + 57 129 51-65
IBT-111 − 58
IBT-112 + 59 130 66-80
IBT-112 − 60
IBT-113 + 61 131 81-95
IBT-113 − 62
IBT-114 + 63 132 96-110
IBT-114 − 64
IBT-115 + 65 133 111-125
IBT-115 − 66
IBT-116 + 67 134 126-140
IBT-116 − 68
IBT-117 + 69 135 141-155
IBT-117 − 70
IBT-118 + 71 136 156-170
IBT-118 − 72
IBT-119 + 73 137 171-185
IBT-119 − 74
IBT-120 + 75 138 186-200
IBT-120 − 76
IBT-121 + 77 139 201-215
IBT-121 − 78
IBT-122 + 79 140 11-25
IBT-122 − 80
IBT-123 + 81 141 26-40
IBT-123 − 82
IBT-124 + 83 142 41-55
IBT-124 − 84
IBT-125 + 85 143 56-70
IBT-125 − 86
IBT-126 + 87 144 71-85
IBT-126 − 88
IBT-127 + 89 145 86-100
IBT-127 − 90
IBT-128 + 91 146 101-115
IBT-128 − 92
IBT-129 + 93 147 116-130
IBT-129 − 94
IBT-130 + 95 148 131-145
IBT-130 − 96
IBT-131 + 97 149 146-160
IBT-131 − 98
IBT-132 + 99 150 161-175
IBT-132 − 100
IBT-133 + 101 151 176-190
IBT-133 − 102
IBT-134 + 103 152 191-205
IBT-134 − 104
IBT-135 + 105 153 206-220
IBT-135 − 106
IBT-158 + 107 154 86-110
IBT-158 − 108
IBT-159 + 109 155 91-110
IBT-159 − 110
aIBT-79 is 13 amino acids in length.
The resulting expression vectors were individually transformed into the arabinose inducible expression strain E. coli BL21-Al (Invitrogen; cat# C6070-03).
Each expression vector was individually transferred into BL21-Al chemically competent E. coli cells for expression analysis. To produce the recombinant protein, 3 mL of LB-ampicillin broth (10 g/L bacto-tryptone, 5 g/L bacto-yeast extract, 10 g/L NaCl, 100 mg/L ampicillin; pH 7.0) was inoculated with one colony of the transformed bacteria and the culture was shaken at 37° C. until the OD600 reached 0.6. Expression was induced by adding 0.03 mL of 20% L-arabinose (final concentration 0.2%, Sigma-Aldrich, St. Louis, Mo.) to the culture and shaking was continued for another 3 hours. For whole cell analysis, 0.1 OD600 mL of cells were collected, pelleted, and 0.06 mL SDS PAGE sample buffer (1×LDS Sample Buffer (Invitrogen cat# NP0007), 6 M urea, 100 mM DTT) was added directly to the whole cells. The samples were heated at 99° C. for 10 minutes to solubilize the proteins. The solubilized proteins were then loaded onto 4-12% gradient MES NuPAGE® gels (NuPAGE® gels cat #NP0322, MES Buffer cat# NP0002; Invitrogen) and visualized with a Coomassie® G-250 stain (SimplyBlue™ SafeStain; Invitrogen; cat# LC6060).
Example 3 Verification of Inclusion Body Formation
To verify that the fusion partner drove expression into insoluble inclusion bodies, it was necessary to lyse the collected cells (0.1 OD600 mL of cells) and fractionate the insoluble from the soluble fraction by centrifugation. Cells were lysed using CelLytic™ Express (Sigma, St. Louis, Mo. cat# C-1990) according to the manufacturer's instructions. Cells that do not produce inclusion bodies undergo complete lysis and yielded a clear solution. Cells expressing inclusion bodies appeared turbid even after complete lysis.
The method used to rank all inclusion body tags was a subjective visual inspection of SimplyBlue™ SafeStain stained PAGE gels. The scoring system was 0, 1, 2 or 3. If no band is detected then a zero score is given. A score of three is given to very heavily stained wide expressed bands. Bands that are weak are scored a one and moderate bands are scored a two. Any score above zero indicated the presence of inclusion bodies (Table 4). Every amino acid has up to three opportunities to be in a tag. Except for the extreme C— and N-terminals of the scanned protein, there are scores per amino acid. These scores are added to give a final activity score for an individual amino acid.
Soluble and insoluble fractions were separated by centrifugation and analyzed by polyacrylamide gel electrophoresis and visualized with SimplyBlue™ SafeStain. Analysis of the cell protein by polyacrylamide gel electrophoresis was used to detect the production of the fusion protein in the whole cell and insoluble fractions but not the soluble cell fraction. Several fusion proteins comprising a 15 amino acid long inclusion body tag derived from amino acid residues 76-175 of SEQ ID NO: 2 were found to be insoluble. This result suggested that it was possible to have very small fusion partners (at least 15 amino acids in length) to facilitate production of peptides in inclusion bodies (Table 4)
Fusion Protein Fusion Protein
Expression Nucleic acid Amino Acid
Plasmid Sequence Sequence
Fusion Protein Designation (SEQ ID NO.) (SEQ ID NO.)
IBT 65-TBP101 pLX240 156 157
IBT 66-TBP101 pLX257 158 159
IBT 67-TBP101 pLX276 160 161
IBT 68-TBP101 pLX242 162 163
IBT 69-TBP101 pLX247 164 165
IBT 70-TBP101 pLX277 166 167
IBT 71-TBP101 pLX241 168 169
IBT 72-TBP101 pLX258 170 171
IBT 73-TBP101 pLX259 172 173
IBT 74-TBP101 pLX260 174 175
IBT 75-TBP101 pLX250 176 177
IBT 76-TBP101 pLX248 178 179
IBT 77-TBP101 pLX244 180 181
IBT 78-TBP101 pLX278 182 183
IBT 79-TBP101 pLX249 184 185
IBT 108-TBP101 pLX266 186 187
IBT 109-TBP101 pLX267 188 189
IBT 110-TBP101 pLX268 190 191
IBT 111-TBP101 pLX269 192 193
IBT 112-TBP101 pLX270 194 195
IBT 113-TBP101 pLX271 196 197
IBT 114-TBP101 pLX272 198 199
IBT 115-TBP101 pLX273 200 201
IBT 116-TBP101 pLX274 202 203
IBT 117-TBP101 pLX275 204 205
IBT 118-TBP101 pLX299 206 207
IBT 119-TBP101 pLX300 208 209
IBT 120-TBP101 pLX301 210 211
IBT 121-TBP101 pLX302 212 213
IBT 122-TBP101 pLX303 214 215
IBT 123-TBP101 pLX304 216 217
IBT 124-TBP101 pLX305 218 219
IBT 125-TBP101 pLX306 220 221
IBT 126-TBP101 pLX307 222 223
IBT 127-TBP101 pLX319 224 225
IBT 128-TBP101 pLX308 226 227
IBT 129-TBP101 pLX235 228 229
IBT 130-TBP101 pLX309 230 231
IBT 131-TBP101 pLX320 232 233
IBT 132-TBP101 pLX310 234 235
IBT 133-TBP101 pLX311 236 237
IBT 134-TBP101 pLX312 238 239
IBT 135-TBP101 pLX321 240 241
IBT 158-TBP101 pLX343 242 243
IBT 159-TBP101 pLX344 244 245
Zein-based Inclusion Body
IBT Tag Amino Acid Sequence Expression
Designation (SEQ ID NO:) Ranking
IBT 65 MRVLLVALALLALAA 0
IBT 66 SATSTHTSGGCGCQP 0
IBT 67 PPPVHLPPPVHLPPP 0
IBT 68 VHLPPPVHLPPPVHL 0
IBT 69 PPPVHLPPPVHVPPP 0
IBT 70 VHLPPPPCHYPTQ 2
IBT 71 RPQPHPQPHPCPCQQ 3
IBT 72 PHPSPCQLQGTCGVG 0
IBT 73 STPILGQCVEFLRHQ 2
IBT 74 CSPTATPYCSPQCQS 0
IBT 75 LRQQCCQQLRQVEPQ 1
IBT 76 HRYQAIFGLVLQSIL 0
IBT 77 QQQPQSGQVAGLLAA 0
IBT 78 QIAQQLTAMCGLQQP 0
IBT 79 TPCPYAAAGGVPH 1
IBT 108 VALALLALAASATST 0
IBT 109 HTSGGCGCQPPPPVH 0
IBT 110 LPPPVHLPPPVHLPP 0
IBT 111 PVHLPPPVHLPPPVH 0
IBT 112 LPPPVHVPPPVHLPP 0
IBT 113 PPCHYPTQPPRPQPH 3
IBT 114 PQPHPCPCQQPHPSP 2
IBT 115 CQLQGTCGVGSTPIL 1
IBT 116 GQCVEFLRHQCSPTA 0
IBT 117 TPYCSPQCQSLRQQC 1
IBT 118 CQQLRQVEPQHRYQA 0
IBT 119 IFGLVLQSILQQQPQ 0
IBT 120 SGQVAGLLAAQIAQQ 0
IBT 121 LTAMCGLQQPTPCPY 0
IBT 122 LALAASATSTHTSGG 0
IBT 123 CGCQPPPPVHLPPPV 0
IBT 124 HLPPPVHLPPPVHLP 0
IBT 125 PPVHLPPPVHLPPPV 0
IBT 126 HVPPPVHLPPPPCHY 0
IBT 127 PTQPPRPQPHPQPHP 3
IBT 128 CPCQQPHPSPCQLQG 0
IBT 129 TCGVGSTPILGQCVE 1
IBT 130 FLRHQCSPTATPYCS 3
IBT 131 PQCQSLRQQCCQQLR 2
IBT 132 QVEPQHRYQAIFGLV 1
IBT 133 LQSILQQQPQSGQVA 0
IBT 134 GLLAAQIAQQLTAMC 0
IBT 135 GLQQPTPCPYAAAGG 0
IBT 158 PTQPPRPQPHPQPHPCPCQQPH 2
IBT 159 RPQPHPQPHPCPCQQPHPSP 2
Example 4 Synthesis, Cloning, and Evaluation of Fusion Peptides Comprising Inclusion Body Tags IBT-180 and IBT-181
The expression ranking data from the various inclusion body tags was evaluated and used to design two additional inclusion body tags (IBT-180 and IBT-181) comprising a T7 translational enhancer (MASMTGGQQMG; SEQ ID NO: 246) linked to the N-terminal portion of an inclusion body forming region of the zein protein. This sequence was provided to standardize the critical N-terminal translated sequence, which is known to be especially important in determining translation initiation efficiency (Stormo, G. “Translation Initiation” in Reznikov, W and Gold, L, Maximizing Gene Expression Butterworths, Boston, Mass. (1986) pp. 195-224.)
Design of Inclusion Body Tags IBT-180 and IBT-181
An alignment of the inclusion body tags exhibiting inclusion body forming ability was performed against the zein protein. The initial library of overlapping inclusion body tags was designed span the entire length of the zein protein. Based on the overlapping nature of the inclusion body tag library, every amino acid had up to three opportunities to be in a tag. Relative scores were assigned to each amino acid within the zein protein based on the frequency of occurrence within a peptide tag capable of inducing inclusion body formation. The relative scores were used to assign a final activity score for each amino acid. When activity score for each amino acid was plotted over the length of the scanned protein, a map was generated depicting the ability of certain domains on the scanned protein to induce inclusion body formation. From this assessment, it was determined that inclusion body tags prepared from the region of the zein protein encompassed by amino acid residues 76-175 of SEQ ID NO: 2 was particularly effective in inducing inclusion body formation.
A 100 amino acid long functional inclusion body tag, IBT-181 (SEQ ID NO: 248), comprising amino acid residues 76 to 175 of SEQ ID NO: 2 and a shorter 30 amino acid inclusion body tag, IBT-180 (SEQ ID NO: 247), comprising a subset of this region (amino acid residues 76 to 105 of SEQ ID NO: 2) were prepared. Both tags also included a short 11 amino acid T7 tag (a translational enhancer) (MASMTGGQQMG; SEQ ID NO: 246) added to the N-terminus of each tag.
Synthesis and Cloning Procedure of IBT-180 and IBT-181
The nucleic acid molecules encoding the inclusion body tags IBT-180 (SEQ ID NO: 247) and IBT-181 (SEQ ID NO: 248) were synthesized and delivered as plasmids harboring kanamycin resistance by DNA 2.0 Inc. (Menlo Park, Calif.). The nucleotide sequence encoding each inclusion body tag was flanked by NdeI and BamHI restriction sites.
The vector comprising the nucleic acid molecule encoding the IBT-180 tag was digested in Buffer 2 (New England Biolabs 10 mM Tris-HCl, 10 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol; pH7.9) with the NdeI and BamHI restriction enzymes (New England Biolabs Beverly, Mass.). Likewise, the test system expression vector pLX121 (SEQ ID NO: 16) was digested with NdeI and BamHI as described in the previous examples. The IBT-180 inclusion body tag restriction digest was directly ligated to the NdeI/BamHI digested test expression vector pLX121 with T4 DNA Ligase (New England Biolabs Beverly, Mass. cat# M0202) at 12° C. for 18 hours. Ampicillin resistant colonies were sequenced. The sequence of the plasmid (pLX363) was confirmed. Expression plasmid pLX363 comprises the chimeric gene encoding the IBT 180-TBP101 fusion protein (SEQ ID NOs: 249 and 250), operably linked to an arabinose inducible promoter.
Inclusion body tag IBT-181 (SEQ ID BO: 248) was cloned using the same procedure as described for IBT-180, resulting in the expression plasmid pLX364. Expression plasmid pLX364 comprises the chimeric gene encoding the IBT 181-TBP101 fusion protein (SEQ ID NOs: 251 and 252), operably linked to an arabinose inducible promoter.
Transformation and Expression of IBT-180 and IBT-181
Expression plasmids pLX363 and pLX364 were transformed, expressed, and evaluated using the procedures described in Examples 2 and 3. The expression ranking results are provided in Table 5.
Inclusion Body Tag Expression Ranking for
IBT-180 and IBT-181
IBT 180 MASMTGGQQMGVHLPPPPCHY 2
PTQPPRPQPHPQPHPCPCQQ
IBT 181 MASMTGGQQMGVHLPPPPCHY 2
PTQPPRPQPHPQPHPCPCQQPH
PSPCQLQGTCGVGSTPILGQCVE
FLRHQCSPTATPYCSPQCQSLR
QQCCQQLRQVEPQHRYQAIFGL
1. An inclusion body tag consisting of 15 contiguous amino acids residues from amino acids residues 76 to 175 of SEQ ID NO: 2.
2. A fusion peptide comprising the inclusion body tag of claim 1 operably linked to at least one peptide of interest.
3. The fusion peptide of claim 2, further comprising at least one cleavable peptide linker having a cleavage site.
4. The fusion peptide of claim 2 wherein the peptide of interest is selected from the group consisting of a peptide that binds to a polymer, a peptide that binds to hair, a peptide that binds to nail, a peptide that binds to skin, a peptide that binds to an antimicrobial substance.
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US (1) US7732569B2 (en)
US20070243198A1 (en) * 2006-02-23 2007-10-18 Heifetz Peter B Production of biologically active proteins
US5110729A (en) 1985-11-14 1992-05-05 Daiichi Seiyaku Co., Ltd. Method of producing peptides using baculovirus vectors in cultured cells
US5670340A (en) 1991-08-19 1997-09-23 Suntory Limited Process for producing peptides in E. coli
WO2003100021A2 (en) 2002-05-24 2003-12-04 Restoragen, Inc. Methods and dna constructs for high yield production of polypeptides
WO2004003207A1 (en) * 2002-06-28 2004-01-08 Era Plantech, S.L. Production of peptides and proteins by accumulation in plant endoplasmic reticulum-derived protein bodies
US6699689B1 (en) 1998-06-09 2004-03-02 Samyang Genex Corporation Mass production method of antimicrobial peptide and DNA construct and expression system thereof
US20040142492A1 (en) 2001-05-10 2004-07-22 Holger Kiesewetter Method for detecting blood cell antigens and the antibodies in response to the same
Chien et. al., The Two-Hybird System: A Method to Identify and Clone Genes for Proteins that Interact With a Protein of Interest, Proc. Natl. Acad. Sci., 1991, vol. 88:9578-9582.
D.J. Kemp, Direct Immunoassay for Detecting Escherichia coli Colonies that Contain Polypeptides Encoded by Cloned DNA Segments, Proc. Natl. Acad. Sci., 1981, vol. 78:4520-4524.
Dykes et. al., Expression of Atrial Natriuretic Factor as a Cleavable Fusion Protein With Chloramphenicol Acetyltransferase in Escherichia coli, Eur. J. Biochem., 1988, vol. 174:411-416.
Gram et. al., A Novel Approach for High Level Production of a Recombinant Human Parathyroid Hormone Fragment in Escherichia coli, Bio/Technology, 1994, vol. 12:1017-1023.
Haught et. al., Recombinat Production and Purification of Novel Antisense Antimicrobial Peptide in Escherichia coli, Biotechnol. Bioengineer., 1988, vol. 57:55-61.
Kempe et. al., Multiple-Copy Genes: Production and Modification of Monomeric Peptides From Large Multimeric Fusion Proteins, Gene, 1985, vol. 39:239-245.
Kuliopulos et. al., Production, Purification, and Cleavage of Tandem Repeats of Recombinant Peptides, J. Am. Chem. Soc., 1994, vol. 116:4599-4607.
Pilon et. al., Ubiquitin Fusion Technology: Bioprocessing of Peptides, Biotechnol. Prog., 1997, vol. 13:374-379.
Ray et. al., Production of Recombinant Salmon Calcitonin by In Vitro Amidation of an Escherichia coli Produced Precursor Peptide, Bio/Technology, 1993, vol. 11:64-70.
Schellenberger et. al., Peptide Production by a Combination of Gene Expression, Chemical Syntheisis, and Protease-Catalyzed Conversion, Int. J. Peptide Protein Res., 1993, vol. 41:326-332.
Shen et. al., Multiple Joined Genes Prevent Product Degradation in Escherichia coli, Proc. Natl. Acad. Sci., 1984, vol. 281:4627-4631.
U.S. Appl. No. 10/935,254, filed Mar. 10, 2005, John P. O'Brien et al.
U.S. Appl. No. 10/935,642, filed Mar. 10, 2005, Xueying Huang et al.
US8163880B2 (en) 2006-02-23 2012-04-24 Era Biotech S.A. Production of biologically active proteins
US20080096246A1 (en) 2008-04-24 application
Mao et al. 2004 Sortase-mediated protein ligation: a new method for protein engineering
Weiner et al. 1998 A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins
Robinson et al. 2004 Tat-dependent protein targeting in prokaryotes and chloroplasts
Tang et al. 2001 Biosynthesis of a highly stable coiled-coil protein containing hexafluoroleucine in an engineered bacterial host
Zettler et al. 2009 The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans‐splicing reaction
Reyes-Turcu et al. 2009 Polyubiquitin binding and disassembly by deubiquitinating enzymes
Sung et al. 1991 Yeast RAD6 encoded ubiquitin conjugating enzyme mediates protein degradation dependent on the N‐end‐recognizing E3 enzyme.
US20030064435A1 (en) 2003-04-03 Compositions and methods for protein secretion
US20050175581A1 (en) 2005-08-11 Biological entities and the pharmaceutical and diagnostic use thereof
Baker et al. 1994 Protein expression using cotranslational fusion and cleavage of ubiquitin. Mutagenesis of the glutathione-binding site of human Pi class glutathione S-transferase.
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