Abstract:
The invention is directed to the enhanced expression and purification of lipoxygenase enzymes. These enzymes are of wide-spread industrial importance, often produced in heterologous microbial systems. Preferably, the lipoxygenase produced by the methods of the invention is a plant-derived enzyme and expressed at high-levels in a microbial system that includes a protease-deficient host and one or more chaperone expression plasmids. The invention is also directed to amino acid and nucleic acid fragments of the lipoxygenase enzyme including fragments in expression constructs encoding all or a portion of one or more lipoxygenase genes. The invention is also directed to methods of manufacturing bread and other food and also non-food products with lipoxygenase manufactured by the methods of the invention.

Description:
REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a continuation of U.S. Non-Provisional application Ser. No. 14/263,105, filed Apr. 28, 2014, which claims priority to U.S. Provisional Application No. 61/817,077, filed Apr. 29, 2013, both of the same title and both of which are incorporated by reference in their entirety. 
     
    
     SEQUENCE LISTING 
       [0002]    The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Apr. 28, 2014, is named 3060.002.US_SL.txt and is 29,528 bytes in size. 
       BACKGROUND 
       [0003]    1. Field of the Invention 
         [0004]    The invention is directed to systems, compositions and methods for the expression and purification of lipoxygenases, to amino acid and nucleic acid sequences of all or portions of lipoxygenases, to molecular constructs for the expression of lipoxygenases, and, in particular, to methods for the large scale production and use of lipoxygenases in food products. 
         [0005]    2. Description of the Background 
         [0006]    Lipoxygenases (LOXs; EC1.13.11_), also known as lipoxydases, are non-heme iron-containing dioxygenases distributed in plants and animals. LOXs catalyze hydroperoxidation of polyunsaturated fatty acids in the first step of fatty acid metabolite synthesis, to produce an unsaturated fatty acid hydroperoxide. A LOX definition according to enzyme classification is linoleate: oxygen oxidoreductase (for plant LOX) and arachidonate: oxygen oxidoreductase (for mammalian LOX). In plants, the most common LOX substrates linoleic acid and linolenic acids are converted into a variety of bioactive mediators involved in plant defense, senescence, seed germination, as well as plant growth and development (Grechkin A. Recent developments in biochemistry of the plant lipoxygenase pathway; Prog Lipid Res. 1998 Nov. 37(5):317-52). Lipoxygenases with different specificities, subcellular location, and tissue-specific expression patterns have been identified as ubiquitously found across kingdoms from bacteria to mammals. 
         [0007]    LOXs are of commercial value in various industries including but not limited to food-related applications in food processing including bread making (bleaching and improved texture), aroma and flavor enhancement as well as for production of perfumes, paint driers (lipoxygenases: potential starting biocatalysts for the synthesis of signaling compounds. Joo Y C, Oh D K. 2012) and pitch control in softwood pulp (Microbial and enzymatic control of pitch in the pulp and paper industry, Ana Gutiérrez &amp; José C. del Río &amp; Angel T. Martínez, Appl Microbiol Biotechnol (2009) 82:1005-1018). Lipoxygenase is present in seeds (e.g. soybeans), grains and many other plant tissues. In the presence of oxygen, lipoxygenase oxidizes unsaturated fatty acids and produces lipid hydroperoxides, which improve dough structure through the oxidation of unsaturated fatty acids and subsequently react with specific chemical components of flour. As a consequence, dough stability and rising is increased, which either or together can increase the volume of the final product. 
         [0008]    Regarding the processing of bread, lipoxygenase enzymes offer an advantage over current chemical additives. The flour ingredient industry had long been using chemical bleach, mostly benzoyl peroxide (BPO). Because of potential health concerns over BPO, some Euro countries and China banned the usage of BPO in flour. In the U.S., BPO is still widely used, but the demand keeps shirking although there is currently no safe alternative. Azodiformamide is another chemical alternative, but the dosage is limited to 40 ppm. At this trace dosage, the bleaching effect is quite restrained. In contrast, enzyme additives especially LOXs can replace chemicals to allow for the processing of flour, resulting in the bleaching of bread and its improved texture. In addition, lipid hydroperoxidases decolorize dough and oxidizes carotinoids, converting them into colorless compounds. This blanching of the dough results in lighter colored product, which is highly desired. 
         [0009]    With regard to enzymes employed in the food industry, regulations frequently require enzymes to be recognized or proven as safe for use. In the case of lipoxygenases, considering that they are ubiquitously found in plants and consumed by humans and animals alike, plant lipoxygenases are considered safe for use, and therefore, of major value to the industry. Although soy extracts containing high levels of lipoxygenases have been used as an additive for bread manufacturing, soy produces an undesirable taste and smell and, accordingly, not often a useful option. 
         [0010]    Because of plant LOX value, many attempts at high-level expression of recombinant plant derived LOX from soy, rice, potato and other sources by heterologous expression in microbial hosts including, but not limited to bacteria such as  E. coli  (BL21 strain),  Bacilli , and in yeast has been attempted, though production was limited [3-8]. The best of these, although still a poor expression from  E. coli , was observed at very cold temperatures [8]. Only one lipoxygenase was produced in  Bacilli  at high-levels (˜160 mg/L), but the lipoxygenase was from a bacterial enzyme, not a plant and consequently not approved for use in the human food industry [9, 10]. In addition, yields still could not achieve desired levels. Accordingly, a need exists for high level expression of plant lipoxygenases that is generally recognized as safe for use in foods, and easily produced in large quantities. 
       SUMMARY OF THE INVENTION 
       [0011]    The present invention overcomes the problems and disadvantages associated with current strategies and designs, and provides new methods and compositions involving the heterologous expression, purification and use of lipoxygenases. 
         [0012]    One embodiment of the invention is directed to the heterologous expression of lipoxygenases in microbes. 
         [0013]    Another embodiment of the invention is directed to methods for the purification of lipoxygenases, preferably from heterologous expression systems according to the invention. 
         [0014]    Another embodiment of the invention is directed to lipoxygenase polypeptide and nucleic acids sequences and molecular constructs of lipoxygenase coding sequences, preferably for the high level expression of lipoxygenase as compared to expression in wild-type host cells. Preferably wild-type host cells are cells that do not contain a protease deficiency and/or cells that do not contain one or more chaperones. 
         [0015]    Another embodiment of the invention is directed to methods for the manufacture of bread products comprising adding lipoxygenases of the invention to a dough containing unsaturated fatty acids and/or carotinoids. Preferably the lipoxygenase reacts with components of the flour forming lipid hydroperoxides increasing the stability of the dough and enhancing the volume of the baked goods. 
         [0016]    Another embodiment of the invention is directed to purified lipoxygenase enzyme made by the methods of the invention. When the purified enzyme is added to dough, another embodiment of the invention comprises products made with the purified enzyme added to dough such as, preferably, bread products. The manufacture of bread products of the invention preferably comprises adding lipoxygenases to a dough containing unsaturated fatty acids and/or carotinoids. Preferably the lipoxygenase reacts with components of the flour forming lipid hydroperoxides increasing the stability of the dough and enhancing the volume of the baked goods. 
         [0017]    Other embodiments and advantages of the invention are set forth in part in the description, which follows, and in part, may be obvious from this description, or may be learned from the practice of the invention. 
     
    
     
       DESCRIPTION OF THE FIGURES 
         [0018]      FIG. 1  Western analysis of SLP1 indicates varying profiles of degradation. M=marker. Different K12 cells with different genotypes are presented in sets of “U” (uninduced) and “I” (induced). 
           [0019]      FIG. 2  Co-expression of the GroEL-GroES chaperone enhances SLP1 production. Four chaperones A-D were co-expressed in  E. coli  with SLP1. Left Panel: SLP1 is directed detected as a weak band in SDS-PAGE as a result of Co-expression with Chaperone B. Right Lane: Enhanced expression of SLP1 with co-expressed GroEL-GroES is confirmed by standard Western analysis. 
           [0020]      FIG. 3  Single step purification of SLP1 from the 424 vector (native protein sequence, no polyhistidine tag). All lanes: SLP1 lacking a his tag was eluted from Zinc-NTA (IMAC) columns and run in SDS PAGE followed by protein staining: Lanes 1—crude lysate; Lane 2—column flow-through; Lane 3—column wash; Lanes 4-7—Zinc-NTA (IMAC) elution with SLP1 loaded in the presence of 0, 2, 5 and 10 mM imidazole, each eluted with 80 mM imidazole. 
           [0021]      FIG. 4   E. coli  cell lines used to verify experiments. 
           [0022]      FIG. 5  SLP1 expression in  E. coli ; SDS PAGE protein gel of whole cell K12  E. coli  lysate expressing SLP1. 
       
    
    
     DESCRIPTION OF THE INVENTION 
       [0023]    Lipoxygenase enzymes (also referred to herein as LOX) are widely used in commercial processing of food products, the manufacture of perfumes and painting products, and in the processing of wood pulp. Although all lipoxygenase catalyze the same basic function, only plant lipoxygenases have been approved by the United States Food and Drug Administration for use in foods and food products. Despite their broad uses, lipoxygenase enzymes are only expressed at low levels and, consequently, commercial quantities are both expensive and difficult to produce. 
         [0024]    Despite previous failures in achieving high level LOX expression, it has been surprisingly discovered that considerable enhancement of plant lipoxygenase expression can be achieved. At least part of this high-level of expression is attributed to the selection of sequences being expressed, expression of the sequences in a protease deficient host, and/or the co-expression with one or more chaperone plasmid sequences. Preferable, the increased expression achieved is at a higher level than expression in host cells that do not contain a protease deficiency and/or cells that do not contain one or more chaperone plasmids. Preferably the expression of the one or more proteases is eliminated, reduced to an undetectable level using conventional detection or reduced by at least 90%, all as compared to wild-type expression levels. 
         [0025]    One embodiment of the invention comprises a system containing a bacterial cell host, preferably with a deficiency or one or more proteases, containing a coding sequence for lipoxygenase enzyme and preferably a chaperone system comprising one or more chaperone molecules. The system is preferably inducible and also preferably maintained from about 10° C. to about 37° C. for a period of time for maximal expression of enzyme product. The period of time is preferably from minutes to hours to days, and more preferably from about 1 to about 24 hours, more preferably from 2 to 12 hours and more preferably from about 2 to about 4 hours. The cells are preferably maintained at temperatures from about 15° C. to about 25° C. during this period. 
         [0026]    The lipoxygenase enzyme may be derived from animal or bacterial cells, and is preferably derived from plant cells. Expression constructs may contain all or a portion of the lipoxygenase gene or coding region. Preferably constructs contain a portion of the coding region sufficient to create functional lipoxygenase activity. Preferably the constructs of the invention encode the sequences of SEQ ID NOs 1-3, or contain the nucleic acid sequences of SEQ ID NOs 4-6. Also preferably the sequence is a functional sequences that generates functional lipoxygenase activity. 
         [0027]    Preferably the host cell is a microorganism that rapidly and economically proliferates in vitro such as, for example, one or more of the bacterial cell strains of K12 cells,  E. coli  cells,  Bacillus  cells,  Lactococci  or yeast cells. Also preferably, the host cells contain one or more protease deficiencies as compared to wild-type cells. For  E. coli  host cells, the deficiency is preferably of one or more of the proteases Lon, OMPT, and/or Lon/ClpP. 
         [0028]    Preferably the host cells further contain one or more chaperone plasmid expression vectors. Chaperones function in assisting protein folding, benefiting the co-expressed molecules. 
         [0029]    Expression of lipoxygenase in the systems of the invention typically involves inducing expression of the lipoxygenase sequence and also preferably the chaperone sequences before, during or after expression of the lipoxygenase, and preferably simultaneously or nearly simultaneously to allow for maximal expression of the enzyme. 
         [0030]    Lipoxygenase produced according to methods of the invention can be further isolated and purified. Preferably, purification of lipoxygenase produced according to the methods of the invention involves contact the with immobilized-metal affinity chromatography media. The enzyme remains bound and can be washed with wash buffer and subsequently eluted with elution buffer. 
         [0031]    Preferably the increased lipoxygenase expression of the invention is 5 fold greater as compared to expression in wild-type cells (e.g., cells that are not protease deficient and/or cells without one or more expression chaperones), more preferably 10 fold greater, more preferably 50 fold greater, more preferably 100 fold greater, more preferably 200 fold greater, more preferably 300 fold greater, more preferably 400 fold greater, and more preferably 500 fold greater or more. 
         [0032]    Lipoxygenase made according to the invention is preferably useful in the manufacture of food products such as bread products (for either, or both bleaching and improving texture), the manufacture of paints thinners, perfumes, aroma and flavor enhancers, as signaling compounds, and for pitch control in softwood pulp in paper industry. 
         [0033]    The following examples illustrate embodiments of the invention, but should not be viewed as limiting the scope of the invention. 
       EXAMPLES 
     Example 1 
     LOXs Employed For Protein Production 
       [0034]    SLP1 (seed linoleate 13S-lipoxygenase-1  [Glycine max ] NCBI Reference Sequence: NP_001236153.1, length 839 amino acids) and SLP3 seed linoleate 9S-lipoxygenase-3 [ Glycine max ] NCBI Reference Sequence: NP_001235383.1) were employed as LOXs for production in microbes. In addition, a shortened version of SLP1 (herein minilox) from amino acid Serine 278 containing an additional methionine before the Serine 278 were cloned and expressed in microbes. 
       Example 2 
     Synthesis of DNA Encoding Protein Sequences for SLP1, SLP3 and Minilox Optimized For Expression 
       [0035]    Optimal gene codon usage in plants and bacteria differ. New DNA encoding sequences for SLP1, SLP3 and minilox were determined and synthetically generated according to instructions (Genscript USA Inc.). The sequence for minilox was identical as that of SLP1 with the exception of having an ATG encoding for an initiator methionine prior to nucleotide bases encoding for SLP1 Serine 278. Optimized sequences with desired cloning sites were created. 
       Example 3 
     Cloning of Soybean Lipoxygenase 1 (SLP1) and 3 (SLP3) and MiniLox 
       [0036]    Initially, SLP1 and SLP3 were cloned into the pET 47b vector (Novogen) using SmaI-XhoI restriction sites, so that each contained the pET47b initiating methionine and a 6X histidine tag (SEQ ID NO 7). The SLP1 and minilox encoding DNA were then transferred to the DNA2.0 expression vector 424 purple, a low copy number plasmid without the histidine tags using NdeI-Xhol sites, so that expressed proteins would not contain the histidine tags. Similarly, the SLP1 encoding sequence was cloned into the 424-purple vector (herein 424 vector) with the exception of using NdeI-EcoRI cloning sites. The SLP1 encoding sequences were then transferred to the DNA2.0 purple-444 vector (herein 444 vector), a high copy number plasmid using restriction sites Ndel-XhoI. The vectors contained promoters for expression of the insert DNA with the pET47 containing a T7-promotor, and the DNA2.0 vectors contained a T5-promotor. 
       Example 4 
     Expression of SLP1, Minilox and SLP3 
       [0037]    Vectors were transfected into cell lines. Initially, expression of SLP1 was performed with the 6 histidine (SEQ ID NO 7) tagged SLP1 vector in  E. coli  BL21 cells, an  E. coli  B cell line suitable for the expression of the pET47b vectors. Thereafter, all expression was performed in  E. coli  K12 strains. Expression was tested in LB media, with 50-100 μg/m1 ampicillin, and induction of expression for all vectors was with 0.5-1 mM IPTG. 
       Example 5 
     Activity Assay 
       [0038]    The activity assay utilized linoleic acid as a substrate and colorimetric detection of product. Detected values for the assay varied depending on the substrate preparation, age of substrate, and substrate batch, which may be subject to variation due to oxidation from the environment. As such, approximate expression levels of SLP1 in BL21 are presented as 1 unit/cell OD550 SLP1-LB culture and relative and approximate values for expression in other strains is relative to the BL21 expression. Cell OD550 is defined as a cell density at OD550. 
       Example 6 
     Improvement of SLP1 Production 
       [0039]    SLP1 expressed with or without a histidine tag using the pET47b vector and BL21 cells was very poor when induced at room temperature. The standard level of activity, 1 unit/OD cells was established for induction at 15° C. with and overnight expression. Dramatically improved activity was observed using the purple-424 vector (herein 424 vector), in the K12 HMS 174 cell line (4 units/Cell OD550). Unlike BL21 cells, activity was also observed when induced at 20° C.-25° C. with overnight expression. Slp1 activity could be further enhanced by growing cells at 15° C. for up to several days. In all  E. coli  strains tested, growth at 37C of was found to generate little or no SLP1 activity, and protein degradation products were observed upon western analysis ( FIG. 2 ). 
         [0040]    An additional increase in activity was discovered using protease deficient  E. coli  K12 strains with the 424 vector. Lon, OMPT, or Lon/ClpP mutants all showed a further minimum two-fold increase in activity with (˜10 units/cell OD550). The specific  E. coli  cell lines with specific protease deficiencies also showed some similar characteristics of protein degradation ( FIG. 3 ) yet some cell lines had less degradation than others, signifying that proteases play a role in the limited production of lipoxygenases. 
         [0041]    An additional enhancement of activity was observed when using the 444 high plasmid copy vector in the K12 Pam155 (lon protease deficient)  E. coli  cell line and with chaperones. Chaperone plasmid sets consisting of five different plasmids from Takara Bio Inc. each designed to express a single or multiple molecular chaperone sets can enable optimal protein expression and folding and reduce protein misfolding. Each Takara plasmid carries an origin of replication (ORF) derived from pACYC and a chloramphenicol-resistance gene (Cmr) gene, which allows the use of  E. coli  expression systems containing ColE1-type plasmids that confer ampicillin resistance. The chaperone genes are situated downstream of the araB or Pzt-1 (tet) promoters and, as a result, expression of target proteins and chaperones can be individually induced if the target gene is placed under the control of different promoters (e.g., lac). These plasmids also contain the necessary regulator (araC or tet r ) for each promoter. Takara Bio Inc. plasmids containing chaperones or sets thereof either tetracycline or arabinose inducible were coexpressed with SLP1. These include: groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, or tig in plasmids (TakaraBo Inc.). Expression of SLP1 in the presence of groES-groEL alone or with tig (groES-groEL-tig) enhanced the amount of active enzyme produced roughly to 40-60 units/cell OD. Activity was optimal at 15° C. but also observed at or below 25° C. At 37° C., expression was more limited. 
         [0042]    Expression of SLP1 in the Pam153 cell with concomitant GroESL chaperone expression, in LB, produces 68 micrograms of SLP1 per milliliter at a bacterial OD550 of 3, when grown in test tubes at 37° C. and induced at 20° C. overnight. However, SLP1 expression in an  E. coli  strain that is not a protease deficient strain and without chaperone expression can either not be detected at all with standard SDS PAGE analysis, or western analysis, or expresses less than 1 μg per milliliter LB under similar conditions at an OD550 of 3 (see  FIG. 4 ). In general, expression of SLP1 in  E. coli  strains grown and induced under optimal conditions was undetectable or less than 1 microgram per milliliter when appropriate chaperones were absent and strains were not protease deficient. However when expressing SLP1 in  E. coli  K12 protease deficient strains with co-expression of an appropriate chaperone, 68 micrograms of SLP1 per milliliter at a bacterial OD550 of 3 was attained. 
       Example 7 
     Purification of SPL1 
       [0043]    Purification of SLP1 with the 6Xhis tag (SEQ ID NO 7) was highly effective using standard Ni-NTA IMAC purification. In the 424 or 444 vectors lacking the 6Xhis tag (SEQ ID NO 7), where SLP1 was encoded by the native SLP1 sequence alone, IMAC was equally efficient though under modified conditions. Nickel and zinc were each tested with similar results and calcium or other divalent metals should do as well. Buffers for IMAC were either 50 mM phosphate or Tris-HCl at pH 7-9, with 400 mM NaCl and 10% glycerol. Cells were disrupted using B-PER (Peirce) or by a homogenizer, in the presence of PMSF as a protease inhibitor. Employing Zinc-NTA, it was discovered that loading the sample in buffer with 10 mM imidazole and elution in buffer with 80 mM imidazole was effective in purification of SLP1. Other column media that effectively binds SLP1 include MonoQ and DEAE, but not negatively charged resins. 
       Example 8 
     Novel Information Provides Improved SLP1 Expression 
       [0044]    Preliminary studies indicate that relatively poor production of SLP1 is the result of rapid proteolysis accompanied by improper folding of the enzyme. The limited soluble SLP1 and lack of insoluble protein suggests that most of the protein produced was rapidly degraded. Degradation products of SLP1 are visible in different  E. coli  strains with different protease deficient genetic backgrounds (see  FIG. 1 ). An increase in both active enzyme and total protein was observed when inducing at suboptimal growth temperatures, where proteases are less functional. A relative increase in production and activity of SLP1 when protein folding is enhanced by an over-expressed chaperone. 
       Example 9 
     High Level Expression of Lipoxygenase in the  E. Coli , K12, 
       [0045]    Unless otherwise stated, all bacterial media employed in this example was Luria Broth (herein LB, consisting of 10 grams Tryptone, 5 grams Yeast Extract, and 10 grams NaCl, dissolved in 1 liter water, and sterilized for a minimum of 20 minutes in an autoclave). Soybean Lipoxygenase 1 (herein SLP1) was expressed from a plasmid transfected into  E. Coli  K12 cells.  FIG. 5  represents an SDS-PAGE protein gel of whole cell soluble proteins extracted from the K12 cells employing the commercial B-PER Protein Extraction Reagent (Pierce, Cat#78243), following company protocols. The highest level of soluble SLP1 protein relative to total soluble protein in the cell extract was 30% or greater and approximated at 34% as estimated by the ImageJ (National Institute of Health public software) analysis software. These levels are consistent with high level production of the enzyme. M=marker, 1 Uninduced, 2 SLP1 induced with 0.5 mM IPTG 3&amp;4 Induced with 0.5 mM IPTG and expressing a molecular chaperone. 
       CITED REFERENCES 
       [0000]    
       
         1. Permiakova, M. D. and V. A. Trufanov, Effect of soybean lipoxygenae on baking properties of wheat flour, Prikl Biokhim Mikrobiol, 2011. 47(3): p. 348-54. 
         2. Permiakova, M. D., et al., [Role of lipoxygenase in the determination of wheat grain quality]. Prikl Biokhim Mikrobiol, 2010. 46(1): p. 96-102. 
         3. Kanamoto, H., M. Takemura, and K. Ohyama, Cloning and expression of three lipoxygenase genes from liverwort,  Marchantia polymorpha  L., in  Escherichia coli . Phytochemistry, 2012. 77: p. 70-8. 
         4. Osipova, E. V., et al., Recombinant maize 9-lipoxygenase: expression, purification, and properties. Biochemistry Biokhimi         , 2010. 75(7): p. 861-5. 
         5. Hwang, I. S. and B. K. Hwang, The pepper 9-lipoxygenase gene CaLOX1 functions in defense and cell death responses to microbial pathogens. Plant Physiol, 2010. 152(2): p. 948-67. 
         6. Padilla, M. N., et al., Functional characterization of two 13-lipoxygenase genes from olive fruit in relation to the biosynthesis of volatile compounds of virgin olive oil. J Agric Food Chem, 2009. 57(19): p. 9097-107. 
         7. Knust, B. and D. von Wettstein, Expression and secretion of pea-seed lipoxygenase isoenzymes in  Saccharomyces cerevisiae . Appl Microbiol Biotechnol, 1992. 37(3): p. 342-51. 
         8. Steczko, J., et al., Effect of ethanol and low-temperature culture on expression of soybean lipoxygenase L-1 in  Escherichia coli . Protein Expr Purif, 1991. 2(2-3): p. 221-7. 
       
     
       Sequence Information 
       [0054]      
         [0000]    
       
         
               
             
           
               
                 SLP1 polypeptide sequence 
               
               
                 (SEQ ID NO 1) 
               
               
                 MFSAGHKIKGTVVLMPKNELEVNPDGSAVDNLNAFLGRSVSLQLISAT 
               
               
                   
               
               
                 KADAHGKGKVGKDTFLEGINTSLPTLGAGESAFNIHFEWDGSMGIPGA 
               
               
                   
               
               
                 FYIKNYMQVEFFLKSLTLEAISNQGTIRFVCNSWVYNTKLYKSVRIFF 
               
               
                   
               
               
                 ANHTYVPSETPAPLVSYREEELKSLRGNGTGERKEYDRIYDYDVYNDL 
               
               
                   
               
               
                 GNPDKSEKLARPVLGGSSTFPYPRRGRTGRGPTVTDPNTEKQGEVFYV 
               
               
                   
               
               
                 PRDENLGHLKSKDALEIGTKSLSQIVQPAFESAFDLKSTPIEFHSFQD 
               
               
                   
               
               
                 VHDLYEGGIKLPRDVISTIIPLPVIKELYRTDGQHILKFPQPHVVQVS 
               
               
                   
               
               
                 QSAWMTDEEFAREMIAGVNPCVIRGLEEFPPKSNLDPAIYGDQSSKIT 
               
               
                   
               
               
                 ADSLDLDGYTMDEALGSRRLFMLDYHDIFMPYVRQINQLNSAKTYATR 
               
               
                   
               
               
                 TILFLREDGTLKPVAIELSLPHSAGDLSAAVSQVVLPAKEGVESTIWL 
               
               
                   
               
               
                 LAKAYVIVNDSCYHQLMSHWLNTHAAMEPFVIATHRHLSVLHPIYKLL 
               
               
                   
               
               
                 TPHYRNNMNINALARQSLINANGIIETTFLPSKYSVEMSSAVYKNWVF 
               
               
                   
               
               
                 TDQALPADLIKRGVAIKDPSTPHGVRLLIEDYPYAADGLEIWAAIKTW 
               
               
                   
               
               
                 VQEYVPLYYARDDDVKNDSELQHWWKEAVEKGHGDLKDKPWWPKLQTL 
               
               
                   
               
               
                 EDLVEVCLIIIWIASALHAAVNFGQYPYGGLIMNRPTASRRLLPEKGT 
               
               
                   
               
               
                 PEYEEMINNHEKAYLRTITSKLPTLISLSVIEILSTHASDEVYLGQRD 
               
               
                   
               
               
                 NPHWTSDSKALQAFQKFGNKLKEIEEKLVRRNNDPSLQGNRLGPVQLP 
               
               
                   
               
               
                 YTLLYPSSEEGLTFRGIPNSISI 
               
               
                   
               
               
                 SLP3 polypeptide sequence 
               
               
                 (SEQ ID NO 2) 
               
               
                 MLGGLLHRGHKIKGTVVLMRKNVLDVNSVTSVGGIIGQGLDLVGSTLD 
               
               
                   
               
               
                 TLTAFLGRSVSLQLISATKADANGKGKLGKATFLEGIITSLPTLGAGQ 
               
               
                   
               
               
                 SAFKINFEWDDGSGIPGAFYIKNFMQTEFFLVSLTLEDIPNHGSIHFV 
               
               
                   
               
               
                 CNSWIYNAKLFKSDRIFFANQTYLPSETPAPLVKYREEELHNLRGDGT 
               
               
                   
               
               
                 GERKEWERIYDYDVYNDLGDPDKGENHARPVLGGNDTFPYPRRGRTGR 
               
               
                   
               
               
                 KPTRKDPNSESRSNDVYLPRDEAFGHLKSSDFLTYGLKSVSQNVLPLL 
               
               
                   
               
               
                 QSAFDLNFTPREFDSFDEVHGLYSGGIKLPTDIISKISPLPVLKEIFR 
               
               
                   
               
               
                 TDGEQALKFPPPKVIQVSKSAWMTDEEFAREMLAGVNPNLIRCLKDFP 
               
               
                   
               
               
                 PRSKLDSQVYGDHTSQITKEHLEPNLEGLTVDEAIQNKRLFLLDHHDP 
               
               
                   
               
               
                 IMPYLRRINATSTKAYATRTILFLKNDGTLRPLAIELSLPHPQGDQSG 
               
               
                   
               
               
                 AFSQVFLPADEGVESSIWLLAKAYVVVNDSCYHQLVSHWLNTHAVVEP 
               
               
                   
               
               
                 FIIATNRHLSVVHPIYKLLHPHYRDTMNINGLARLSLVNDGGVIEQTF 
               
               
                   
               
               
                 LWGRYSVEMSAVVYKDWVFTDQALPADLIKRGMAIEDPSCPHGIRLVI 
               
               
                   
               
               
                 EDYPYTVDGLEIWDAIKTWVHEYVFLYYKSDDTLREDPELQACWKELV 
               
               
                   
               
               
                 EVGHGDKKNEPWWPKMQTREELVEACAIIIWTASALHAAVNFGQYPYG 
               
               
                   
               
               
                 GLILNRPTLSRRFMPEKGSAEYEELRKNPQKAYLKTITPKFQTLIDLS 
               
               
                   
               
               
                 VIEILSRHASDEVYLGERDNPNWTSDTRALEAFKRFGNKLAQIENKLS 
               
               
                   
               
               
                 ERNNDEKLRNRCGPVQMPYTLLLPSSKEGLTFRGIPNSISI 
               
               
                   
               
               
                 Minilox1 polypeptide sequence 
               
               
                 (SEQ ID NO 3) 
               
               
                 MSTPIEFHSFQDVHDLYEGGIKLPRDVISTIIPLPVIKELYRTDGQHI 
               
               
                   
               
               
                 LKFPQPHVVQVSQSAWMTDEEFAREMIAGVNPCVIRGLEEFPPKSNLD 
               
               
                   
               
               
                 PAIYGDQSSKITADSLDLDGYTMDEALGSRRLFMLDYHDIFMPYVRQI 
               
               
                   
               
               
                 NQLNSAKTYATRTILFLREDGTLKPVAIELSLPHSAGDLSAAVSQVVL 
               
               
                   
               
               
                 PAKEGVESTIWLLAKAYVIVNDSCYHQLMSHWLNTHAAMEPFVIATHR 
               
               
                   
               
               
                 HLSVLHPIYKLLTPHYRNNMNINALARQSLINANGIIETTFLPSKYSV 
               
               
                   
               
               
                 EMSSAVYKNWVFTDQALPADLIKRGVAIKDPSTPHGVRLLIEDYPYAA 
               
               
                   
               
               
                 DGLEIWAAIKTWVQEYVPLYYARDDDVKNDSELQHWWKEAVEKGHGDL 
               
               
                   
               
               
                 KDKPWWPKLQTLEDLVEVCLIIIWIASALHAAVNFGQYPYGGLIMNRP 
               
               
                   
               
               
                 TASRRLLPEKGTPEYEEMINNHEKAYLRTITSKLPTLISLSVIEILST 
               
               
                   
               
               
                 HASDEVYLGQRDNPHWTSDSKALQAFQKFGNKLKEIEEKLVRRNNDPS 
               
               
                   
               
               
                 LQGNRLGPVQLPYTLLYPSSEEGLTFRGIPNSISI 
               
               
                   
               
               
                 SLP1 DNA optimized encoding sequence (with 
               
               
                 restriction sites 5′ SmaI and 3′ XhoI with stop 
               
               
                 codon for cloning into pET47b with 6X histidine 
               
               
                 tag (SEQ ID NO 7)) 
               
               
                 (SEQ ID NO 4) 
               
               
                 CCCGGGATGTTTAGTGCTGGTCACAAAATCAAAGGTACCGTGGTCCTG 
               
               
                   
               
               
                 ATGCCGAAAAATGAACTGGAAGTCAACCCGGATGGTAGCGCCGTTGAT 
               
               
                   
               
               
                 AACCTGAATGCGTTCCTGGGTCGTAGCGTGTCTCTGCAGCTGATTTCC 
               
               
                   
               
               
                 GCCACCAAAGCAGACGCTCACGGCAAGGGTAAAGTTGGCAAAGATACG 
               
               
                   
               
               
                 TTTCTGGAAGGTATTAATACCTCCCTGCCGACCCTGGGTGCCGGTGAA 
               
               
                   
               
               
                 TCAGCTTTCAACATCCATTTCGAATGGGATGGTTCAATGGGCATTCCG 
               
               
                   
               
               
                 GGCGCCTTCTACATCAAAAACTACATGCAGGTGGAATTTTTCCTGAAA 
               
               
                   
               
               
                 AGTCTGACCCTGGAAGCAATCTCCAATCAGGGTACGATTCGTTTTGTC 
               
               
                   
               
               
                 TGCAACTCGTGGGTGTATAATACCAAACTGTACAAAAGCGTTCGCATC 
               
               
                   
               
               
                 TTTTTCGCGAACCACACCTATGTTCCGAGCGAAACCCCGGCACCGCTG 
               
               
                   
               
               
                 GTTTCTTACCGTGAAGAAGAACTGAAAAGTCTGCGCGGCAATGGTACC 
               
               
                   
               
               
                 GGCGAACGTAAAGAATATGATCGCATTTATGACTACGATGTTTACAAC 
               
               
                   
               
               
                 GACCTGGGCAATCCGGATAAAAGCGAAAAACTGGCCCGTCCGGTCCTG 
               
               
                   
               
               
                 GGCGGTAGCTCTACCTTCCCGTATCCGCGTCGCGGTCGTACCGGTCGT 
               
               
                   
               
               
                 GGTCCGACCGTGACCGATCCGAACACCGAAAAACAGGGCGAAGTCTTT 
               
               
                   
               
               
                 TATGTGCCGCGCGACGAAAATCTGGGCCATCTGAAATCTAAAGATGCC 
               
               
                   
               
               
                 CTGGAAATCGGTACCAAAAGTCTGTCCCAGATTGTGCAACCGGCGTTT 
               
               
                   
               
               
                 GAAAGCGCCTTCGATCTGAAATCTACGCCGATTGAATTTCACTCCTTC 
               
               
                   
               
               
                 CAGGACGTTCATGATCTGTATGAAGGCGGTATCAAACTGCCGCGTGAC 
               
               
                   
               
               
                 GTCATTTCAACCATTATCCCGCTGCCGGTGATCAAAGAACTGTACCGC 
               
               
                   
               
               
                 ACGGATGGTCAGCACATTCTGAAATTTCCGCAACCGCATGTGGTTCAG 
               
               
                   
               
               
                 GTTTCACAATCGGCGTGGATGACCGATGAAGAATTCGCGCGTGAAATG 
               
               
                   
               
               
                 ATCGCCGGCGTTAACCCGTGCGTCATTCGCGGTCTGGAAGAATTTCCG 
               
               
                   
               
               
                 CCGAAAAGCAATCTGGACCCGGCAATCTATGGCGATCAGAGTTCCAAA 
               
               
                   
               
               
                 ATTACCGCTGACTCTCTGGACCTGGATGGCTACACGATGGATGAAGCC 
               
               
                   
               
               
                 CTGGGTAGTCGTCGCCTGTTTATGCTGGACTATCACGATATCTTCATG 
               
               
                   
               
               
                 CCGTACGTGCGTCAGATTAACCAACTGAATTCTGCAAAAACCTATGCT 
               
               
                   
               
               
                 ACCCGTACGATCCTGTTTCTGCGCGAAGACGGCACGCTGAAACCGGTT 
               
               
                   
               
               
                 GCAATTGAACTGAGCCTGCCGCATTCTGCTGGTGATCTGAGTGCCGCG 
               
               
                   
               
               
                 GTGTCCCAGGTTGTGCTGCCGGCAAAAGAAGGCGTTGAAAGTACCATC 
               
               
                   
               
               
                 TGGCTGCTGGCGAAAGCCTATGTTATTGTCAACGATTCATGTTACCAT 
               
               
                   
               
               
                 CAACTGATGTCGCACTGGCTGAATACCCATGCAGCTATGGAACCGTTT 
               
               
                   
               
               
                 GTTATCGCAACGCATCGCCACCTGTCTGTCCTGCACCCGATTTATAAA 
               
               
                   
               
               
                 CTGCTGACCCCGCATTACCGTAACAATATGAACATCAATGCACTGGCT 
               
               
                   
               
               
                 CGCCAGAGTCTGATTAACGCGAATGGTATTATCGAAACCACGTTCCTG 
               
               
                   
               
               
                 CCGTCAAAATATTCGGTGGAAATGTCATCGGCCGTTTACAAAAACTGG 
               
               
                   
               
               
                 GTCTTTACCGACCAGGCACTGCCGGCTGATCTGATCAAACGTGGCGTC 
               
               
                   
               
               
                 GCGATTAAAGATCCGAGCACCCCGCATGGTGTGCGTCTGCTGATTGAA 
               
               
                   
               
               
                 GACTATCCGTACGCGGCCGATGGCCTGGAAATCTGGGCAGCTATTAAA 
               
               
                   
               
               
                 ACCTGGGTGCAGGAATATGTTCCGCTGTATTACGCACGCGATGACGAT 
               
               
                   
               
               
                 GTGAAAAATGACTCCGAACTGCAACACTGGTGGAAAGAAGCTGTTGAA 
               
               
                   
               
               
                 AAAGGTCATGGCGACCTGAAAGATAAACCGTGGTGGCCGAAACTGCAG 
               
               
                   
               
               
                 ACCCTGGAAGATCTGGTGGAAGTTTGTCTGATTATCATTTGGATTGCC 
               
               
                   
               
               
                 AGCGCACTGCATGCCGCGGTGAACTTTGGTCAATATCCGTACGGCGGT 
               
               
                   
               
               
                 CTGATTATGAATCGTCCGACCGCAAGCCGTCGCCTGCTGCCGGAAAAA 
               
               
                   
               
               
                 GGCACGCCGGAATACGAAGAAATGATCAACAACCATGAAAAAGCGTAC 
               
               
                   
               
               
                 CTGCGCACCATCACGAGCAAACTGCCGACCCTGATTAGCCTGTCTGTT 
               
               
                   
               
               
                 ATCGAAATTCTGTCAACGCACGCGTCGGATGAAGTCTATCTGGGTCAG 
               
               
                   
               
               
                 CGTGACAACCCGCATTGGACCAGTGATTCCAAAGCGCTGCAGGCCTTC 
               
               
                   
               
               
                 CAAAAATTCGGCAACAAACTGAAAGAAATCGAAGAAAAACTGGTCCGT 
               
               
                   
               
               
                 CGCAACAATGATCCGAGCCTGCAGGGTAACCGTCTGGGTCCGGTGCAA 
               
               
                   
               
               
                 CTGCCGTATACCCTGCTGTATCCGTCCAGTGAAGAAGGTCTGACGTTT 
               
               
                   
               
               
                 CGTGGTATTCCGAACTCCATTTCCATCTGACTCGAG 
               
               
                   
               
               
                 SLP3 DNA optimized encoding sequence (with 
               
               
                 restriction sites 5′ NdeI and 3′ EcoRI and 3′ 
               
               
                 stop codon for cloning into the pJex purple 424 
               
               
                 vector from DNA2.0 Inc. 
               
               
                 (SEQ ID NO 5) 
               
               
                   CATATG CTGGGCGGCCTGCTGCACCGTGGTCATAAAATCAAGGGCACC 
               
               
                   
               
               
                 GTGGTCCTGATGCGTAAGAACGTCCTGGATGTGAATAGCGTGACCTCG 
               
               
                   
               
               
                 GTCGGCGGTATTATCGGCCAGGGTCTGGACCTGGTGGGTAGCACGCTG 
               
               
                   
               
               
                 GATACCCTGACGGCCTTTCTGGGCCGCTCAGTGTCGCTGCAACTGATC 
               
               
                   
               
               
                 AGCGCAACCAAAGCAGATGCTAACGGCAAAGGCAAGCTGGGCAAGGCG 
               
               
                   
               
               
                 ACGTTCCTGGAAGGCATTATCACCTCCCTGCCGACGCTGGGTGCAGGC 
               
               
                   
               
               
                 CAGTCAGCCTTTAAAATTAATTTCGAATGGGATGACGGCTCTGGTATT 
               
               
                   
               
               
                 CCGGGCGCCTTCTACATCAAGAACTTCATGCAGACCGAATTTTTCCTG 
               
               
                   
               
               
                 GTCAGCCTGACGCTGGAAGATATCCCGAATCATGGCTCGATTCACTTT 
               
               
                   
               
               
                 GTGTGCAACAGCTGGATCTACAATGCGAAACTGTTCAAGTCCGATCGC 
               
               
                   
               
               
                 ATTTTCTTTGCCAATCAGACCTATCTGCCGTCAGAAACGCCGGCACCG 
               
               
                   
               
               
                 CTGGTTAAATACCGTGAAGAAGAACTGCACAACCTGCGTGGTGACGGT 
               
               
                   
               
               
                 ACCGGTGAACGTAAAGAATGGGAACGCATCTACGATTACGACGTTTAC 
               
               
                   
               
               
                 AACGATCTGGGTGATCCGGACAAAGGCGAAAACCATGCGCGTCCGGTC 
               
               
                   
               
               
                 CTGGGCGGTAATGACACCTTTCCGTACCCGCGTCGCGGTCGTACCGGT 
               
               
                   
               
               
                 CGTAAACCGACGCGTAAGGATCCGAACAGCGAATCTCGCAGTAATGAT 
               
               
                   
               
               
                 GTGTATCTGCCGCGTGACGAAGCCTTTGGTCACCTGAAAAGCTCTGAT 
               
               
                   
               
               
                 TTCCTGACGTACGGCCTGAAGTCCGTTTCACAGAACGTCCTGCCGCTG 
               
               
                   
               
               
                 CTGCAAAGCGCATTTGATCTGAATTTCACCCCGCGCGAATTTGATTCG 
               
               
                   
               
               
                 TTCGACGAAGTTCATGGTCTGTATAGCGGCGGTATTAAGCTGCCGACC 
               
               
                   
               
               
                 GACATTATCTCTAAAATCAGTCCGCTGCCGGTGCTGAAGGAAATTTTT 
               
               
                   
               
               
                 CGCACGGATGGCGAACAGGCTCTGAAGTTCCCGCCGCCGAAAGTCATC 
               
               
                   
               
               
                 CAAGTGTCGAAAAGCGCGTGGATGACCGATGAAGAATTTGCACGTGAA 
               
               
                   
               
               
                 ATGCTGGCTGGTGTTAACCCGAATCTGATTCGCTGTCTGAAGGATTTC 
               
               
                   
               
               
                 CCGCCGCGTTCCAAACTGGATTCACAGGTGTATGGTGACCACACCAGT 
               
               
                   
               
               
                 CAAATCACGAAAGAACATCTGGAACCGAACCTGGAAGGCCTGACCGTT 
               
               
                   
               
               
                 GATGAAGCTATTCAGAATAAACGTCTGTTTCTGCTGGATCATCACGAC 
               
               
                   
               
               
                 CCGATCATGCCGTATCTGCGTCGCATTAATGCGACCTCGACGAAAGCG 
               
               
                   
               
               
                 TACGCCACCCGCACGATCCTGTTCCTGAAGAACGATGGTACCCTGCGT 
               
               
                   
               
               
                 CCGCTGGCCATTGAACTGAGCCTGCCGCATCCGCAGGGTGACCAATCG 
               
               
                   
               
               
                 GGTGCGTTTAGCCAGGTTTTCCTGCCGGCCGATGAAGGCGTCGAAAGT 
               
               
                   
               
               
                 TCCATCTGGCTGCTGGCAAAAGCTTATGTGGTTGTCAACGATTCTTGC 
               
               
                   
               
               
                 TACCATCAGCTGGTGTCTCACTGGCTGAATACCCATGCAGTGGTTGAA 
               
               
                   
               
               
                 CCGTTTATTATCGCTACGAACCGCCACCTGTCTGTCGTGCATCCGATC 
               
               
                   
               
               
                 TATAAACTGCTGCATCCGCACTACCGCGACACCATGAACATTAATGGT 
               
               
                   
               
               
                 CTGGCGCGTCTGAGTCTGGTCAACGATGGCGGTGTGATTGAACAGACG 
               
               
                   
               
               
                 TTTCTGTGGGGCCGTTATTCTGTTGAAATGAGTGCCGTTGTCTACAAA 
               
               
                   
               
               
                 GATTGGGTCTTCACCGACCAAGCACTGCCGGCAGACCTGATCAAGCGT 
               
               
                   
               
               
                 GGTATGGCAATTGAAGATCCGTCCTGTCCGCACGGCATCCGTCTGGTG 
               
               
                   
               
               
                 ATTGAAGATTATCCGTACACCGTTGACGGTCTGGAAATCTGGGATGCA 
               
               
                   
               
               
                 ATTAAAACGTGGGTGCATGAATACGTTTTTCTGTACTACAAGTCTGAT 
               
               
                   
               
               
                 GACACCCTGCGCGAAGACCCGGAACTGCAGGCGTGCTGGAAAGAACTG 
               
               
                   
               
               
                 GTGGAAGTTGGTCACGGCGATAAAAAGAACGAACCGTGGTGGCCGAAA 
               
               
                   
               
               
                 ATGCAAACCCGTGAAGAACTGGTTGAAGCGTGTGCCATTATCATTTGG 
               
               
                   
               
               
                 ACGGCAAGCGCTCTGCATGCGGCCGTGAACTTTGGCCAGTATCCGTAC 
               
               
                   
               
               
                 GGCGGTCTGATTCTGAATCGCCCGACCCTGTCTCGTCGCTTCATGCCG 
               
               
                   
               
               
                 GAAAAAGGCAGTGCTGAATATGAAGAACTGCGTAAAAATCCGCAGAAG 
               
               
                   
               
               
                 GCGTACCTGAAAACCATCACGCCGAAATTTCAAACCCTGATTGACCTG 
               
               
                   
               
               
                 AGCGTGATCGAAATTCTGTCCCGCCATGCGTCAGATGAAGTTTATCTG 
               
               
                   
               
               
                 GGTGAACGTGACAACCCGAATTGGACCTCCGATACGCGTGCACTGGAA 
               
               
                   
               
               
                 GCTTTTAAGCGCTTCGGCAACAAACTGGCCCAGATCGAAAACAAGCTG 
               
               
                   
               
               
                 TCAGAACGTAACAACGATGAAAAGCTGCGTAATCGCTGCGGCCCGGTG 
               
               
                   
               
               
                 CAAATGCCGTATACCCTGCTGCTGCCGTCCTCAAAAGAAGGTCTGACG 
               
               
                   
               
               
                 TTCCGTGGTATCCCGAATAGCATTAGCATC TAAGAATTC   
               
               
                   
               
               
                 Minilox optimized encoding sequence (with 5′ 
               
               
                 NdeI and 3′ XhoI restriction sites and 3′ stop 
               
               
                 codon for cloning into pJexpress purple 424 
               
               
                 vector from DNA2.0 Inc.) 
               
               
                 (SEQ ID NO 6) 
               
               
                 CATATGTCTACGCCGATTGAATTTCACTCCTTCCAGGACGTTCATGAT 
               
               
                   
               
               
                 CTGTATGAAGGCGGTATCAAACTGCCGCGTGACGTCATTTCAACCATT 
               
               
                   
               
               
                 ATCCCGCTGCCGGTGATCAAAGAACTGTACCGCACGGATGGTCAGCAC 
               
               
                   
               
               
                 ATTCTGAAATTTCCGCAACCGCATGTGGTTCAGGTTTCACAATCGGCG 
               
               
                   
               
               
                 TGGATGACCGATGAAGAATTCGCGCGTGAAATGATCGCCGGCGTTAAC 
               
               
                   
               
               
                 CCGTGCGTCATTCGCGGTCTGGAAGAATTTCCGCCGAAAAGCAATCTG 
               
               
                   
               
               
                 GACCCGGCAATCTATGGCGATCAGAGTTCCAAAATTACCGCTGACTCT 
               
               
                   
               
               
                 CTGGACCTGGATGGCTACACGATGGATGAAGCCCTGGGTAGTCGTCGC 
               
               
                   
               
               
                 CTGTTTATGCTGGACTATCACGATATCTTCATGCCGTACGTGCGTCAG 
               
               
                   
               
               
                 ATTAACCAACTGAATTCTGCAAAAACCTATGCTACCCGTACGATCCTG 
               
               
                   
               
               
                 TTTCTGCGCGAAGACGGCACGCTGAAACCGGTTGCAATTGAACTGAGC 
               
               
                   
               
               
                 CTGCCGCATTCTGCTGGTGATCTGAGTGCCGCGGTGTCCCAGGTTGTG 
               
               
                   
               
               
                 CTGCCGGCAAAAGAAGGCGTTGAAAGTACCATCTGGCTGCTGGCGAAA 
               
               
                   
               
               
                 GCCTATGTTATTGTCAACGATTCATGTTACCATCAACTGATGTCGCAC 
               
               
                   
               
               
                 TGGCTGAATACCCATGCAGCTATGGAACCGTTTGTTATCGCAACGCAT 
               
               
                   
               
               
                 CGCCACCTGTCTGTCCTGCACCCGATTTATAAACTGCTGACCCCGCAT 
               
               
                   
               
               
                 TACCGTAACAATATGAACATCAATGCACTGGCTCGCCAGAGTCTGATT 
               
               
                   
               
               
                 AACGCGAATGGTATTATCGAAACCACGTTCCTGCCGTCAAAATATTCG 
               
               
                   
               
               
                 GTGGAAATGTCATCGGCCGTTTACAAAAACTGGGTCTTTACCGACCAG 
               
               
                   
               
               
                 GCACTGCCGGCTGATCTGATCAAACGTGGCGTCGCGATTAAAGATCCG 
               
               
                   
               
               
                 AGCACCCCGCATGGTGTGCGTCTGCTGATTGAAGACTATCCGTACGCG 
               
               
                   
               
               
                 GCCGATGGCCTGGAAATCTGGGCAGCTATTAAAACCTGGGTGCAGGAA 
               
               
                   
               
               
                 TATGTTCCGCTGTATTACGCACGCGATGACGATGTGAAAAATGACTCC 
               
               
                   
               
               
                 GAACTGCAACACTGGTGGAAAGAAGCTGTTGAAAAAGGTCATGGCGAC 
               
               
                   
               
               
                 CTGAAAGATAAACCGTGGTGGCCGAAACTGCAGACCCTGGAAGATCTG 
               
               
                   
               
               
                 GTGGAAGTTTGTCTGATTATCATTTGGATTGCCAGCGCACTGCATGCC 
               
               
                   
               
               
                 GCGGTGAACTTTGGTCAATATCCGTACGGCGGTCTGATTATGAATCGT 
               
               
                   
               
               
                 CCGACCGCAAGCCGTCGCCTGCTGCCGGAAAAAGGCACGCCGGAATAC 
               
               
                   
               
               
                 GAAGAAATGATCAACAACCATGAAAAAGCGTACCTGCGCACCATCACG 
               
               
                   
               
               
                 AGCAAACTGCCGACCCTGATTAGCCTGTCTGTTATCGAAATTCTGTCA 
               
               
                   
               
               
                 ACGCACGCGTCGGATGAAGTCTATCTGGGTCAGCGTGACAACCCGCAT 
               
               
                   
               
               
                 TGGACCAGTGATTCCAAAGCGCTGCAGGCCTTCCAAAAATTCGGCAAC 
               
               
                   
               
               
                 AAACTGAAAGAAATCGAAGAAAAACTGGTCCGTCGCAACAATGATCCG 
               
               
                   
               
               
                 AGCCTGCAGGGTAACCGTCTGGGTCCGGTGCAACTGCCGTATACCCTG 
               
               
                   
               
               
                 CTGTATCCGTCCAGTGAAGAAGGTCTGACGTTTCGTGGTATTCCGAAC 
               
               
                   
               
               
                 TCCATTTCCATCTGACTCGAG 
               
             
          
         
       
     
         [0055]    Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. The term comprising, where ever used, is intended to include the terms consisting and consisting essentially of. Furthermore, the terms comprising, including, and containing are not intended to be limiting. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims.