JP2008501364A - Expression system comprising functionally linked RGG gene and GTFG promoter - Google Patents

Abstract

  Plasmid-based expression systems that can be used to enhance production of C-repeat regions (CRR) by Streptococcus gordonii, other Streptococcus strains, and other bacteria are provided. This system utilizes the promoter and secretory signal sequence of gtfG combined with the positive regulatory gene rgg to drive the synthesis and secretion of CRR from the resident E. coli or Streptococcus shuttle plasmid pVA838. This plasmid isolated from E. coli is S. coli. Easily introduced to Gordoni, under optimal conditions, the concentration of secreted protein was increased almost 10-fold compared to production by the surface protein expression system (SPEX).

Description

(See sequence listing)
The accompanying sequence listing is fully incorporated herein by reference.

  Expression systems based on Escherichia coli are widely used for the production of heterologous protein products. However, these expression systems are limited to the production of specific proteins that are sensitive to degradation by E. coli proteolytic enzymes, expressed in inclusion bodies, or produced at unacceptably low levels. Possesses usefulness. Thus, there remains a need to produce recombinant proteins that maintain the natural biological activity of the protein and are readily available in large quantities.

  Among other aspects, novel expression systems in which resident plasmids are utilized to enhance the expression of heterologous proteins and polypeptides are provided in the present invention. In one example, the heterologous antigen BH4XCRR is S. It is expressed in gordonii. When using this system, for example, the regulatory gene rgg is utilized to induce the expression of BH4XCRR, and secretion is facilitated by fusion with the GTF secretion signal sequence of the BH4XCRR structural gene. Thus, the product of the rgg gene (regulatory gene) in turn regulates the regulated gene GTF. Also provided is a known composition medium that allows protein production and facilitates its purification.

  The PLEX system has been found useful for the expression of the complete protein product BH4XCRR, which is largely degraded when expressed in E. coli (see Example 1). PLEX is a versatile system for the production of other heterologous protein products that are difficult to express by E. coli. Since PLEX is compatible with SPEX expression systems, both systems can be utilized in a single clone to express individual proteins or multiple proteins. PLEX also serves as a useful tool for the expression of heterologous antigens on the surface of Streptococcus gordonii, which can then be used, for example, as a vaccine vector. Other bacterial species and strains can also be used.

  Nucleic acids comprising an operably linked rgg gene, a gtfG promoter, and a nucleic acid encoding a polypeptide of interest are provided in the present invention; the polypeptide of interest, when synthesized, and the GTF of interest It is a fusion protein of signal sequence. The polypeptide of interest can further comprise the amino acid sequence LPXTG (SEQ ID NO: 24) at the carboxy terminus of the polypeptide of interest. The gene, gtfG promoter, and / or GTF signal sequence is preferably derived from Streptococcus but may be derived from another bacterial strain. Preferred bacteria are S. cerevisiae. Gordoni. Polypeptides of interest include antigens, antibodies, immunogenic fragments of antibodies, enzymes, or any other protein described herein, and may include small molecules peptides and pharmaceutical agents, It is not limited to these. Preferred polypeptides of interest are those designated BH4XCRR and other polypeptides that can be used in immunogenic pharmaceutical compositions (eg, vaccines). Preferably, the regulatory gene (rgg gene) is derived from the same organism as the GTF signal sequence. For example, both the rgg gene and the GTF signal sequence are It will be derived from Gordonii or other Streptococcus. However, it is also contemplated that the regulatory gene rgg gene of one species can be adapted, for example, to the closely related GTF signal sequence of other bacterial species.

  When used in an immunogenic pharmaceutical composition, preferably the composition contains a pharmaceutically acceptable carrier, excipient, or the like. When in the form of a pharmaceutical composition intended to enhance the immune response, the composition may further comprise an adjuvant.

  The portion of the nucleic acid that encodes the polypeptide of interest can further include a second sequence that encodes the purified polypeptide. Examples include, but are not limited to, proteins that assist purification, such as glutathione-s-transferase (GST) and hemagglutinin (HA). Preferably, the purified polypeptide comprises a cleavable domain so that the protein of interest can be cleaved from the purified polypeptide. Other sequences that can be used to facilitate purification and / or detection include, but are not limited to, a 6X-histidine tag and a Flag tag that will be fused to the carboxy terminus of the BH4XCRR sequence. .

  Another embodiment has the nucleic acid of interest functionally incorporated into a vector. These vectors may further comprise a suitable replicon, such as Escherichia and / or Streptococcus replicons. Preferred replicons may provide higher plasmid copy numbers, for example to enhance the amount of recoverable DNA from E. coli. Increasing the plasmid copy number correspondingly increases the amount of heterologous product as a result of maximizing the number of production-related genes in each cell.

The vector may further comprise one or more polylinkers and / or antibiotic selection genes. Preferred polylinkers are inserted between the KpnI and EcoRI restriction sites to facilitate cloning of heterologous genes and / or fusion genes into plasmids that may have one or more KpnI and / or EcoRI restriction sites. Will be done. The vector is capable of replication and polypeptide expression in the host cell. Suitable antibiotic genes include chloramphenicol (erythromycin if not already present), kanamycin (eg, neomycin), tetracycline (tet ^R ), ampicillin (Amp ^R ), and / or carbenicillin. Can be.

  Another aspect of the invention has an expressible vector transformed or transfected in a host cell. Preferred organisms for transformation include Escherichia, Streptococcus, Lactococcus, Lactobacillus bacteria, in particular E. coli, S. cerevisiae. Gordoni, L. Lactis and Lactobacillus species are included. In further embodiments, the host cell can be co-transformed or co-transfected with a surface protein expression (SPEX) vector. Co-transformed / co-transfected refers to a host cell into which a vector comprising a SPEX vector and a PLEX vector has been inserted simultaneously, or a host into which one vector has been inserted first, followed by a second vector. Means a cell.

  Another embodiment contemplates purification of the polypeptide of interest. The polypeptide can be purified from the supernatant of cultured host cells. Alternatively, if the polypeptide of interest expresses the LPXTG (SEQ ID NO: 24) anchoring motif, the polypeptide of interest can be collected in combination with the cell wall for use as an antigen. Preferably, the polypeptide of interest is a substantially purified polypeptide. Substantially purified means that when the protein is separated via PAGE, only the protein of interest is observed after Coomassie staining of the gel.

Furthermore, another aspect is
(A) functionally linking the nucleic acid of claim 1 encoding the polypeptide of interest to a vector;
(B) inserting the vector into a host cell;
(C) culturing a host cell that expresses the nucleic acid under conditions under which the polypeptide of interest is synthesized; and (d) isolating and purifying the polypeptide of interest in the host cell. A method for synthesizing peptides is contemplated.

  The method may use any host cell, but preferably the host cell is a Streptococcus or Escherichia, Streptococcus, Lactococcus, and Lactobacillus host cell. The method may further comprise co-transforming or co-transfecting the host cell with a surface protein expression (SPEX) vector comprising a second polypeptide of interest.

  The drawings are exemplary only and should not be construed as limiting the invention.

  The plasmid-borne expression (PLEX) system is a useful technique for the production of heterologous protein products. This is accomplished by inserting nucleic acids expressing specific endogenous and heterologous genes and the polypeptide of interest into a suitable plasmid vector; Transformed into Gordonii. Both the particular inserted sequences that facilitate production and their placement on resident plasmids distinguish the present invention from previously established expression systems.

  During growth using typical in vitro culture conditions, Gordonii secretes a 172 kDa glucosyltransferase (GTF) into the culture supernatant. The positive regulatory gene rgg is Identified in Gordoni. When rgg is overexpressed using a plasmid complementation method, the rgg gene enhances GTF activity in the culture supernatant.

  Three genetic factors--rgg, a portion of gtfG, and a heterologous gene sequence are required for production and secretion of the heterologous gene product of interest. The expression of the heterologous gene product is driven by an endogenous promoter for gtfG. Expression from the gtfG promoter is positively regulated by Rgg. Product secretion is facilitated by fusion with a heterologous product GTF signal sequence.

  These genetic elements were inserted into the appropriate plasmid for the purposes of the present invention. The plasmid chosen for this role is pVA838, publicly available from the American Type Culture Collection (ATCC). The features that make this plasmid suitable for the present application include the cloning site for insertion of genetic material, the presence of antibiotic resistance markers for detection of transformants, and the presence of both E. coli and Streptococcus replicons. included. Alternative plasmid vectors can also be utilized. Factors containing additional cloning sites (polylinker), cassettes for color selection, antibiotic resistance genes (eg ampicillin, chloramphenicol), different polymerase promoters (eg T7 and T4), and purification Additional genetic factors including, but not limited to, nucleic acid for additional fusion protein sequences that allow rapid column purification and subsequent cleavage of the protein used for It may be inserted into a vector or cassette containing the genetic factor.

  The PLEX system can be readily utilized with standard molecular biology techniques and traditional microbiological laboratory equipment and reagents.

  The PLEX system possesses an enhanced level in the expression of the model protein BH4XCRR compared to the surface protein expression (SPEX) system, which is the most comparable known expression system. The SPEX system is described in detail in US Pat. No. 5,821,088, the contents of which are hereby fully incorporated by reference for all purposes. The PLEX expression system was estimated to secrete approximately 10 times the amount of model product BH4XCRR into the culture supernatant when bacteria were cultured using previously identified optimal growth conditions compared to SPEX. . The concentration of BH4XCRR in the culture supernatant when using the PLEX system was estimated to be 10 mg / L by comparison with the previously purified BH4XCRR standard concentration.

  The PLEX system further overcomes certain limitations associated with conventional E. coli expression systems. Conventional E. coli expression systems relate to the ability to express specific gene products, including products expressed in inclusion bodies, products that are substrates for E. coli proteolytic enzymes, or those that cannot exhibit high levels of expression by bacteria. There is a limit. In addition, problems arise when protein products expressed by E. coli are to be used immunologically, since small amounts of endotoxins that are unintentionally co-purified with the protein product of interest can complicate research. The use of the PLEX system eliminates these problems because the protein is produced by gram positive bacteria that do not contain endotoxin.

  The application of the PLEX system is Bacterial symbiotic vectors such as Gordonii can be used to easily expand to include antigen expression. S. A symbiotic vector such as Gordoni can be used to generate an immunological response to surface expressed heterologous antigens. When PLEX is used, the addition of LPXTG (SEQ ID NO: 24) anchoring motif to the C-terminus of the heterologous product results in the heterologous protein not being secreted, but S. cerevisiae. It can be immobilized on the surface of a symbiotic vector such as Gordoni. In addition, S.M. By transforming a symbiotic vector such as Gordoni with a plurality of PLEX-based plasmids, a plurality of antigens can be expressed as either secreted or immobilized molecules.

  Also described herein are materials and methods for using E. coli expression vectors to generate Streptococcus subunit vaccine candidates named BH4XCRR. BH4XCRR is an approximately 45 kDa antigen composed of four C-repeat regions from the Streptococcus pyogenes M6 protein (FIG. 1). These attempts have shown that BH4XCRR is sensitive to degradation when expressed in E. coli. However, it is possible to use Gram-positive S. cerevisiae using the surface protein expression (SPEX) system. It can be expressed and secreted in complete form by Gordonii.

  When SPEX is used, S.P. A region homologous to the Gordonii chromosome, and A heterologous structural gene such as bh4xcrr is cloned into the plasmid pSMB104 (functionally linked) so that the region corresponding to the Pimmones-derived emm6.1 gene is adjacent to the structural gene (Myscofski et al., 1998 Protein Expression and Purification 14: 409-417). The homologous flanking region promotes chromosomal integration of the structural gene downstream of the P2 promoter. Fusion of the heterologous gene with 16 amino acids (aa) in the N-terminal region of the Piogenes M6 protein provides a secretory signal sequence. SPEX systems are S. Derivatives of Piogenes M6 protein (Myscofski et al., 2000 Protein Expression & Purification 20: 112-123), mouse cytokines IFN-γ and IL-2 (Byrd et al., 2002 Vaccine 20: 2197-2205), and staphylococci It has been used successfully for the production of numerous heterologous proteins, including nuclease A (Dutton et al., 2000 Protein Expression & Purification 19: 158-172). However, the yield of BH4XCRR from the SPEX system was insufficient for the intended application due to, for example, low protein expression and inefficient protein recovery methods.

  In typical in vitro culture conditions, ie, growth using Todd Hewitt broth (THY) or Brain Heart Infusion (BHI) broth containing yeast extract. Gordonii secretes the 172 kDa enzyme glucosyltransferase (GTF) into the culture supernatant. This enzyme synthesizes glucan polymers from sucrose that can play an important role in the formation of microbial biofilms on tooth surfaces (Vickerman et al., 2002 Infection & Immunity 70: 1703-1714; and Vickerman et al. 1997 Applied & Environmental Microbiol. 63: 1667-1673). Mutagenesis of the gene rgg located immediately upstream of gtfG resulted in significantly reduced GTF activity, suggesting a possible regulatory role for rgg (Sulavik et al., 1992 J). Bacteriol. 174: 3577-3586). Investigations on regulatory mechanisms suggest that Rgg may act to enhance both gtfG transcription and translation (Vickerman et al., 1997 Applied & Environmental Microbiol. 63: 1667-1673). The structural prediction of Rgg suggests the presence of a helix-turn-helix motif that suggests a DNA binding protein (Bateman et al., 2000 Nuc. Acids Res. 28: 399-406). Interaction between the sequence located in the 3 ′ region of Rgg and rgg can promote transcriptional activity in the GTF promoter (Sulavik et al., 1992 J. Bacteriol. 174: 3577-3586; and Vickerman et al. , 2003 Microbiology 149: 399-406). Rgg may be able to enhance translation of GTF by melting the predicted hairpin-loop secondary structure sequestering the gtfG ribosome binding site on multicistronic rgg / gtfG mRNA (Sulavik et al., 1992 J. Bacteriol. 174: 3577-3586). S. using plasmid complementation. When overexpressed in Gordoni, Rgg enhanced the level of GTF activity in the culture supernatant approximately 6-fold (Sulavik et al., 1992).

  The PLEX system utilizes Rgg interactions that enhance activity in the gtfG promoter as a means of expressing the heterologous polypeptide of interest. PLEX is an S.I. It can be used by Gordonii to produce secreted or surface-immobilized heterologous products. Using this system, the regulatory gene rgg is integrated into the plasmid in order to induce the expression of the structural gene of interest from the gtfG promoter. Product secretion is facilitated by fusion of the structural gene with the GTF secretion signal sequence. Also described are combinations of unique culture conditions that facilitate rapid and efficient purification of the secreted product. S. Expression of heterologous products on the surface of Gordonii This is accomplished by fusing an anchoring sequence derived from the Piogenes M protein with a heterologous product. In addition, S.M. Described herein is the use of the PLEX system for the expression of multiple heterologous antigens on the surface of a Gordoni.

  The expression system described herein is used to express any polypeptide of interest derived from a pathogen, including enzymes, antigens for use in the preparation of immunogenic preparations, signaling proteins, etc. obtain. Preferably, its expression is a Streptococcus strain, especially S. cerevisiae. Performed in Gordoni. Preferred proteins of interest are proteins that are secreted or expressed on the surface of the organism from which they are derived.

Expression systems can also be utilized to express proteins directly in the culture medium for easy collection of recombinantly produced proteins from the culture medium. Proteins of interest include antigens, enzymes, surface-expressed proteins, eukaryotic proteins, polypeptide fragments, pharmaceuticals, antibodies (antibody fragments such as scFv, Fab, F (ab ′) ₂ ), and immunogens Sex polypeptides may be included.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The following terms are provided below.

  By “substantially purified” is meant a purified polypeptide or nucleic acid in which 80% or more of the polypeptide or nucleic acid is identical. For example, a substantially purified polypeptide will not have other polypeptide contaminants synthesized from another nucleic acid.

Abbreviations and definitions as provided herein are accepted in the art unless otherwise indicated. As used herein, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a patient” includes a plurality of patients, and reference to “the dosage” includes one or more dosages and is known to those skilled in the art. Includes references to their equivalents, etc.
Abbreviation Ab Antibody BHI Brain Heart Infusion Broth CAA Casamino acid CDM Known Composition Medium Cm ^R Chloramphenicol Resistance CRR C-Repeat Region CSP Competence Stimulating Peptide CV Column Capacity dH ₂ O Distilled Water ELISA Enzyme Binding Immunosorbent assay GST Glutathione-s-transferase GTF Glucosyltransferase HA Hemagglutinin IFN-γ Interferon gamma IgG Immunoglobulin G (gamma)
IL-2 Interleukin-2
kbp kilobase pair kDa kilodalton LB Luria-Bertani broth mAb monoclonal antibody PCR polymerase chain reaction PLEX plasmid-derived expression system PVDF polyvinylidene fluoride (membrane)
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SPEX Surface protein expression system THY Todd Hewitt broth containing yeast extract
(Example)

  Although the invention has been described in detail with reference to the following examples, it will be understood that various modifications can be made without departing from the spirit of the compositions and methods disclosed herein.

Secretion of heterologous products The functionality of the PLEX system was tested by expressing an approximately 45 kDa protein product (denoted BH4XCRR) modeled in the M-protein C-repeat region (CRR) from Piogenes. FIG. 2 details the arrangement and alignment of the rgg, gtfG, and bh4xcrr coding sequences when placed on a plasmid to facilitate production of BH4XCRR protein.
Bacterial strains and growth conditions. E. coli strains were grown in Luria-Bertani (LB) broth (Difco, Detroit, MI) or on LB agar (Difco). S. Gordonii was grown in Brain Heart Infusion (BHI) (Difco) or on BHI containing 1.5% agar. S. Gordoni is a known composition medium (CDM) containing 5% casamino acids (CAA) (Fisher Scientific, Pittsburgh, PA) (JRH Biosciences, Lenexa, KS). ). Selection and growth of strains carrying antibiotic resistance determinants was 25 μg chloramphenicol / mL for E. coli strains and S. cerevisiae. For gordonii, 5 μg erythromycin / mL was used.

  DNA isolation and manipulation. Standard molecular biology techniques were followed in DNA preparation and manipulation. The PCR product was cloned into pCR2.1 TOPO (Invitrogen (Carlsbad CA)) and transformed into E. coli InvαF '(Invitrogen) using the manufacturer's procedure. The plasmid was isolated from the E. coli strain using the Qiagen purification kit (Valencia, CA). After digestion with endonuclease, the DNA fragments were separated by electrophoresis and eluted from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen). All restriction endonucleases were obtained from New England Biolabs (Beverely, MA).

  Plasmid pVA838. The E. coli Streptococcus shuttle plasmid pVA838 has been previously characterized (Macrina et al., 1982 Gene 19: 345-353) and was isolated from E. coli obtained from the American Type Culture Collection (Manassas, VA). pVA838 confers resistance to chloramphenicol and erythromycin when transformed into E. coli. S. In Gordoni, pVA838 provides only erythromycin resistance.

  Amplification of rgg / gtfG. Primers (forward primer TW3 5'-CCGGGATCCCGTGTGACGGAGATAGTAG-3 '(SEQ ID NO: 4) and reverse primer TW4 5'-CCGGGGACCCCCTGAACAGCGACTACTTC-3' (SEQ ID NO: 5)) were added to S. cerevisiae. Designed for amplification of a 1.3 kbp product using the Goldis Chalice chromosome as a template. These primers were designed based on the sequence published in rgg and the 5 'region of gtfG (GenBank accession number: M89776). TW3 introduced a BamHI restriction site at the 5 'end of the rgg sequence. PCR using TW3 and reverse primer generated a KpnI restriction site at the 3 'end of the gtfG sequence.

  BH4XCRR design and amplification. BH4XCRR is an S.A. It contains four identical domains modeled in the conserved C-repeat region (CRR) of the M. protein from Piogenes (Figure 1). Each CRR segment contains 70 amino acids. There is a 7 amino acid spacer between the N-terminus of the molecule and between each CRR segment, each having a different amino acid sequence, but suitable for maintaining the alpha-helical coiled-coil nature of the molecule. (Phillips et al., 1981 Proc. Nat'l. Acad. Sci. USA 78: 4689-4693). A 6-residue histidine tag was included at the C-terminus with the initial intention to extend the purification options. Further, the nucleotide sequence of each CRR segment was modified as much as possible while maintaining the same amino acid sequence of each segment. Finally, the frequency of codon usage Optimized for expression in Gordoni. The gene was synthesized by Blue Heron Biotechnology (Bothell, WA) and supplied as an insert in the purified plasmid. In order to amplify the bh4xcrr structural gene using this plasmid as a template, the forward primer TW5 (5′-CCCGGTACCCTGAAACAGAAGGTTAGACGAGGGG-3 ′, SEQ ID NO: 6) and the reverse primer TW6 (5′-ACATGGCATTATGATGGTGTGTGTGTGTGTGTG PCR was performed using No. 7). Amplification of bh4xcrr was performed so that a KpnI restriction site was introduced at the 5 'end with two additional nucleotides to facilitate in-frame fusion with the gtfG secretion signal sequence. PCR amplification was also used to introduce a SphI restriction site and stop codon at the 3 'end of bh4xcrr.

  Construction of pLEX1.0: bh4xcrr. For cloning, pCR2.1 containing the rgg / gtfG insert was serially digested first with KpnI and then with BamHI. Digestion with KpnI was performed at 37 ° C. for 5 hours using the reagents recommended by the manufacturer. Sodium chloride was adjusted to 50 mM and BamHI was added to the reaction for an additional 5 hours. DNA fragments were separated using agarose gel electrophoresis, the 1.3 kbp band was excised, extracted from the agarose matrix and stored at −20 ° C. until further use.

  The bh4xcrr coding sequence was excised from pCR2.1 using simultaneous double digestion with SphI and KpnI according to the manufacturer's protocol for 3 hours at 37 ° C. The 1 kbp DNA fragment was separated using electrophoresis. This band was cut out, extracted from the agarose matrix and stored at -20 ° C for later use.

  Plasmid pVA838 was digested simultaneously at 37 ° C. for 4 hours using BamHI and SphI restriction enzymes according to the reaction conditions recommended by the manufacturer. An approximately 10 kbp DNA fragment was excised from the gel after electrophoresis, extracted, and stored at -20 ° C.

  The bh4xcrr and rgg / gtfG fragments were cloned into pVA838 digested with BamHI and SphI in a triple ligation reaction. Digested DNA was added to the reaction in a quantitative ratio of 5: 5: 1 bh4xcrr: rgg / gtfG: pVA838. Ligation was achieved by overnight incubation at 13 ° C. with T4 DNA ligase (Promega (Madison, Wis.)) Using the reaction conditions indicated by the manufacturer. The ligation product was immediately transformed into the InvαF 'strain of E. coli. Transformants carrying pVA838: rgg / gtfG / bh4xcrr were selected from chloramphenicol resistant colonies. Further selection was based on the isolation of an appropriately sized 11.5 kb plasmid and PCR using the isolated plasmid as a template and primers TW3 and TW6. The DNA sequence of the insert was determined by sequencing primers TW7 (5′-GCAACAGAAATATCACCCTCCCG-3 ′; SEQ ID NO: 8), TW8 (5′-CTTGTGGCTCCAAAGGCCTGTG-3 ′; SEQ ID NO: 9), and TW9 (5′-GGACCCTGCCCACTACTGACCGC-3 '; Confirmed using SEQ ID NO: 10). The plasmid construct was named pLEX1.0: bh4xcrr (see FIG. 2; SEQ ID NO: 1).

  Purified pLEX1.0: bh4xcrr was transformed into S. cerevisiae using a previously established transformation method (Pozzi et al., 1990 Res. Microbiol. 141: 659-670). It was introduced to Gordoni GP251. Briefly, S.I. Gordonii was grown in BHI in the presence of 10% fetal calf serum to induce competence, divided into 450 μl aliquots and stored at −80 ° C. Plasmid pLEX1.0: bh4xcrr was incubated with freshly thawed competent cells at 37 ° C. for 2 hours. This culture was mixed with liquid BHI agar containing 5% defibrinated sheep blood and overlaid on BHI agar. After condensing this layer for a short time, liquid BHI agar containing 5 μg erythromycin / mL was carefully poured into the plate. Colonies selected after 48 hours of growth at 37 ° C. were streaked on BHI agar containing 5 μg erythromycin / mL for further confirmation of resistance and for selection of pure clones. Transformants were confirmed based on detection using a Western blot of BH4XCRR in culture supernatant after overnight growth in BHI containing 5 μg erythromycin / mL at 37 ° C.

  Electrophoresis and Western blot. S. Proteins in Gordoni culture supernatant were separated using SDS-PAGE. Gels were stained using Coomassie Brilliant Blue or transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 3% bovine serum albumin (BSA). The antibody was diluted with a buffer containing 0.5% Tween 20, 0.02% sodium azide, 0.5M sodium chloride, and 0.01M Tris (pH 8.2). Membranes were prepared at a concentration of 2 μg / mL S.M. MAb 10F5 (Jones et al, 1986 J. Exp. Med. 164: 1226-1238) specific for the epitope found in the M-protein C-repeat region of Piogenes and 1: 1000 dilution of goat anti-mouse IgG AP conjugate The antibody (BioRad (Hercules, CA)) was used to probe. Blots were diluted with buffer containing 0.06M Tris, 0.02% sodium azide, 0.06% magnesium chloride hexahydrate (pH 9.8) with AP conjugate substrate kit (BioRad). Developed using.

S. After Gordoni transformation, erythromycin resistant colonies were grown overnight at 37 ° C. in BHI broth. All cultures were back-diluted with CDM / CAA containing erythromycin until reaching stationary phase (OD ₆₅₀ = 0.45 after 1: 5 dilution with dH ₂ O) The optical density of each culture was monitored. Culture supernatants were prepared for SDS-PAGE and BH4XCRR was visualized by Western blot (FIG. 3). Samples were cultured from SPEX strains secreting BH4XCRR and processed to allow comparison of the production levels of these two expression systems. pLEX1.0: bh4xcrr transformation Complete BH4XCRR was detected in 8 of 10 Gordonii culture supernatants, and production in all transformants was enhanced compared to production in the SPEX strain (FIG. 3). Single pLEX1.0: bh4xcrr transformation A Gordoni culture was maintained and designated SRL44.

  BH4XCRR could be readily detected in the culture supernatant using a Western blot probed with mAb 10F5, but the enzyme-linked immunosorbent assay (ELISA) was used to determine the BH4XCRR concentration in the culture supernatant. A number of approaches for quantification have been unsuccessful. Alternatively, Western blots were used to compare the BH4XCRR band intensity in the SRL44 culture supernatant with that of serially diluted, previously purified, BH4XCRR produced by the SPEX system (FIG. 4). Band intensities were quantified using densitometry, and a regression line was calculated from the values. From this evaluation, it was determined that BH4XCRR was secreted into the medium at a concentration of 10 mg / L by SRL44.

Production and purification of heterologous product BH4XCRR A frozen stock of SRL44 (−80 ° C.) was used to inoculate 10 mL of BHI containing 5 μg erythromycin / mL and the culture was incubated at 37 ° C. overnight. It is back-diluted with 1 L of filter sterilized CDM supplemented with 5% CAA and 5 μg erythromycin / mL, and OD ₆₅₀ = 0.45 (1: 5 dilution with dH ₂ O; dH ₂ in a static 37 ° C. incubator; Until it was subtracted from the blank with O). The cells were pelleted by centrifugation and the supernatant was filtered using a filter unit with a 0.45 μm cutoff. In preparation for hydrophobic interaction chromatography, (NH ₄ ) ₂ SO ₄ was added to the supernatant at a concentration of 1.5 M and the supernatant was filtered again to remove the precipitate. Stripped with 5 column volumes (CV) of 20 mM Na ₂ HPO ₄ (pH 7.0) and equilibrated with 5 CV of 50 mM Na ₂ HPO ₄ /1.5 M (NH ₄ ) ₂ SO ₄ (pH 7.0) The supernatant was loaded onto a 5 mL HiTrap Butyl FF column (Amersham BioSciences (Piscataway, NJ)). After the sample was loaded, the column was washed with 10 CV 50 mM Na ₂ HPO ₄ /1.5 M (NH ₄ ) ₂ SO ₄ (pH 7.0). The protein was eluted using a 10 CV (NH ₄ ) ₂ SO ₄ gradient from 1.5 to 0 M and 2 mL fractions were collected. All steps were performed at a flow rate of 5 mL / min. The effectiveness of this approach was assessed by running fractions on an SDS-PAGE gel and the protein was visualized by staining with Coomassie Brilliant Blue (FIG. 5). BH4XCRR was not detected in the pass-through sample taken when the sample was almost completely loaded onto the column, suggesting that the product was efficiently extracted from the supernatant. BH4XCRR eluted from the column in a limited number of fractions and was represented as a single darkly stained 45 kDa band, from which it remained largely intact during this processing step Was suggested. Furthermore, BH4XCRR is S. cerevisiae in gradient elution. Isolated from a number of other proteins secreted by Gordonii.

After this primary cleanup, BH4XCRR was further purified using gel filtration chromatography. Fractions from hydrophobic interaction separations containing BH4XCRR were pooled and proteins were precipitated using a 60% saturated solution of (NH ₄ ) ₂ SO ₄ . The precipitate was pelleted, redissolved in 50 mM citric acid (pH 2.8) and loaded onto an equilibrated HiPrep 16/60 Sephacryl S-300 column (Amersham). BH4XCRR was visualized on a Coomassie-stained SDS-PAGE gel in fractions that eluted 40-75 minutes after sample loading, but four 5 mL collected primarily between 50-65 minutes. It eluted in the fraction (Figure 6). These fractions contained undetectable amounts of contaminating proteins. In the fractions eluted at 60 and 65 minutes, a weak banding pattern was observed below the main BH4XCRR band. This band generation pattern implies the degradation of trace amounts of BH4XCRR, and the reactivity of these bands was confirmed using Western blots probed with mAb 10F5.

The method used for purification of BH4XCRR from 1 L of SRL44 culture supernatant is provided in Table 1 below. The steps are performed in the displayed order.

Expression of Fixed Heterologous Products Using the PLEX Plasmid Using genetics associated with the SPEX system, S. cerevisiae containing a gram-positive anchoring motif. The heterologous gene product is transformed into S. cerevisiae by fusing the C-terminal region from Piogenes M-protein with a structural heterologous gene. It can be fixed to the cell wall of Gordoni (Myscofski et al., 1998 Protein Expression & Purification 14: 409-17). By incorporating this anchoring sequence into the PLEX expression plasmid, the vaccinia virus antigen A27L was transformed into S. cerevisiae using the PLEX expression system. Expressed on the surface of Gordoni.

  pLEX1.1: Construction of bh4xcrr. Site-specific mutation of the EcoRI site located within the 3 'region of rgg to promote the use of additional EcoRI sites on the plasmid for cloning purposes while retaining the functionality of the rgg / gtfG sequence To prepare plasmid pLEX1.1: bh4xcrr. Site-directed mutagenesis was performed using the QuickChange XL site-directed mutagenesis kit (Stratagene (La JoIIa, CA)) according to the manufacturer's protocol. PLEX1.0 as template: bh4xcrr and primers EcoRI-F (5′-CAAAGAAACAATTTGAGCGAATCCAACTAACAGGT-3 ′; SEQ ID NO: 11) and EcoRI-R (5′-TCTGCAACTACTGTTAGTTTGGTTCGCTCAAAT-3 ′; SEQ ID NO: 12) A silent T860C nucleotide mutation was introduced into rgg. The parent plasmid was digested using DpnI and the product was transformed into E. coli XL-10 Gold cells (Stratagene). Erythromycin resistant clones were screened for the presence of the mutation by confirming that EcoRI was unable to excise the band during the digestion reaction. This plasmid was then sequenced to further confirm the mutation. The plasmid construct was named pLEX1.1: bh4xcrr.

  pLEX1.1: Construction of a27l (a). The gene encoding the vaccinia virus Copenhagen A27L (p14) protein was expressed as primers 5'-CGGGGTACCGACGGAACTCTTTTCCC-3 '(SEQ ID NO: 13) and 5'-CCGGGAATTCCCTCATATGATCTGACAC-3' (SEQ ID NO: 14) (underlined sequence) (Represented by restriction sites for KpnI and EcoRI incorporated into the primers, respectively) and vaccinia virus Copenhagen DNA as a template. The PCR product was subcloned into pCR2.1 (Invitrogen) to form plasmid pCR2.1: A27L. Subsequent digestion of plasmid pCR2.1: A27L with the restriction enzymes EcoRI and KpnI yielded a 286 bp fragment that was ligated with the SPEX cloning vector pSMB104 digested with EcoRI and KpnI. Such ligation results in the a27l structural gene lacking a stop codon being transformed into S. cerevisiae. It was positioned in frame immediately upstream of the anchoring region from the Piogenes M6 protein. This fixed type a27l gene was named a27l (a). The DNA sequence of the region encoding A27L of pSMB104: a271 (a) was confirmed by sequencing. pSMB104: Forward primer LB37 (5′-CCGGGGTACCTGACGGAACTCTTTTC-3 ′; SEQ ID NO: 15) and reverse primer ML11 (5′-CGGCCGTCGGATTTAGTTTCTTCTTTTGCGTTTA-3 ′) using a27l (a) as a template; Was used to generate a PCR product carrying the a271 (a) gene flanked by KpnI and NruI restriction sites. This product was digested with KpnI and NruI and the 1.0 kbp band was isolated. To excise the bh4xcrr gene, pLEX1.1: bh4xcrr was digested with KpnI and NruI. The 9.8 kbp band isolated from this reaction was ligated with a 1.0 kbp KpnI and NruI digested PCR product and the sequence of the inserted region was confirmed by DNA sequencing. The sequence of this plasmid, named pLEX1.1: a271 (a) (SEQ ID NO: 2).

  S. Gordonii (GP251 strain) was transformed with pLEX1.1: a271 (a) using the synthetic competence stimulating peptide (CSP) N-DVRSNKIRLWWENIFFNK-COOH (SEQ ID NO: 25). Briefly, GP251 was grown overnight in BHI containing 5 μg chloramphenicol / mL. To prepare cells for transformation, CSP (10 μg / mL final concentration) and glycerol (10% final concentration) were added to the culture, and the cells were divided into 100 μL aliquots and frozen at −80 ° C. S. To transform Gordonii, cells were thawed rapidly and 1 μg DNA and 900 μL THY were added. Cells were incubated for 3 hours at 37 ° C. and seeded on BHI agar containing 5 μg erythromycin / mL.

After selection of erythromycin resistant transformants, S. The presence of A27L on the surface of Gordoni was evaluated. S. Gordoni strains GP251, SRL21 (expressing A27L on the surface using the SPEX system), and pLEX1.1: a27l (a) transformed clone (named SRL45) were OD ₆₅₀ = 0.5 Was grown until. Cells were incubated with anti-vaccinia virus rabbit serum. Cells were then labeled using FITC-conjugated anti-rabbit antibody (Ab) and the fluorescence intensity of 10,000 particles was quantified using a Beckman Coulter FC500 flow cytometer (FIG. 7). Parent S. Gordoni strain GP251 showed a low background level of fluorescence (mean fluorescence channel (MFC) = 2.3). A substantial shift to a larger fluorescence channel was observed for both SRL21 (MFC = 13.3) and SRL45 (MFC = 23.2). These findings suggest that the viral antigen A27L was successfully expressed and immobilized on the surface of SRL45 using the PLEX system.

  Consideration. Since multiple heterologous products have been successfully produced using E. coli, the creation of streptococcal vaccine candidate BH4XCRR was first attempted using this bacterium. Although BH4XCRR can be expressed using E. coli, we have observed substantial degradation of the product. This example highlights the limitations associated with the use of E. coli as an expression vector. Attempts to produce specific products using E. coli can result in poor expression, insoluble protein expression in inclusion bodies, or a lack of ability to express the product in intact form. An additional problem due to the possible presence of trace amounts of lipopolysaccharide (LPS) arises from using E. coli as an expression vector. LPS is a potent toxin and immunostimulant that can be unintentionally co-purified with the desired product.

  Gram-positive bacteria Gordoni is a member of S.G., such as BH4XCRR. It has been used to express a number of heterologous products, including derivatives of the Piogenes M protein. When the SPEX system is used, the heterologous sequence is integrated into the chromosome and production is driven from the native P2 promoter. The secretion is This is accomplished by fusion of the product with a secretory signal sequence derived from the emm6.1 gene from Piogenes. The SPEX system was very successful in producing specific antigens, and the first attempt to make BH4XCRR was particularly promising with respect to complete product expression. However, the yield of BH4XCRR was not sufficient for the intended application. Therefore, PLEX was developed as an enhanced expression system.

  What helped the approach was S. It was an observation that Gordonii secretes certain high molecular weight proteins under a variety of culture conditions. This protein was regularly detected as a major protein band in SDS-PAGE analysis of the culture supernatant. N-terminal sequence analysis revealed that the protein was a 172 kDa glucosyltransferase or GTF. GTF is positively regulated by Rgg at both transcriptional and translational levels. Indeed, overexpression of Rgg using plasmid complement upregulates GTF activity almost 6-fold.

  The expression system described herein was designed to drive the expression of BH4XCRR or other proteins or polypeptides of interest from the gtfG promoter. In order to enhance activity at the gtfG promoter and enhance translation, the coding region of the gtfG positive regulator Rgg was included. Secretion of the product was achieved by fusion of the GTF secretion signal sequence with the bh4xcrr structural gene. BH4XCRR was selected for expression from the plasmid with the expectation that multiple copies of these genes would facilitate enhanced production. S. Gordonii was easily transformed with plasmid pVA838: rgg / gtfG / bh4xcrr and complete BH4XCRR expression by the transformants was confirmed using Western blot. After growth of transformant SRL44 in CDM supplemented with 5% CAA, secreted product was detected in the culture supernatant at a concentration of 10 mg / L. Growth of SRL44 in this medium facilitates rapid purification using a two-step process involving gel filtration followed by hydrophobic interaction chromatography. Minimal degradation of BH4XCRR was observed after this purification procedure.

S. Anchoring multiple heterogeneous products to the surface of Gordoni Surface expression of vaccinia virus antigen B5R by Gordonii. To express the truncated vaccinia virus antigen B5R using the SPEX system, the SRL39 strain was transformed into Created from Gordoni GP251 strain. Forward primer LB7 (5'-CCGGGTTACCATGACTGTACCCACTATGAATAAC (SEQ ID NO: 17), underlined region encodes KpnI restriction site) and reverse primer LB9 (5'-CCGGTCCGACTGCTCTCATAACATTTCATTTC (SEQ ID NO: 18); underlined region A PCR product containing the coding sequence of B5R amino acids 22-276 was generated using vaccinia virus Copenhagen DNA as a template, as well as encoding a SalI restriction site. This product was subcloned into pCR2.1 to generate pCR2.1: B5RΔ, which was digested with KpnI and SalI. A 768 bp fragment was isolated and ligated with pSMB104 digested with KpnI and SalI to generate pSRL39. The sequence of B5RΔ was confirmed by sequencing. S. Gordonii GP251 strain was transformed with pSRL39. Transformants were screened based on erythromycin resistance and PCR was performed to confirm the presence of the B5RΔ gene construct. A single clone was retained and named SRL39.

To assess the surface expression of B5R, GP251 and SRL39 were grown until OD ₆₅₀ = 0.5. After incubation with rabbit anti-vaccinia virus serum, the adsorbed rabbit antibody was detected using FITC-conjugated anti-rabbit IgG. The fluorescence associated with 10,000 particles was recorded and the results are presented in FIG. We observed a substantial shift to a higher fluorescence channel for the SRL39 sample compared to GP251. These findings are consistent with expectations, and B5R This suggests that it is expressed on the surface of Gordonii SRL39 strain.

Construction of pLEX1.3: a271 (a). S. In order to express a plurality of heterologous antigens on the surface of Gordonii, S. cerevisiae expressing vaccinia virus protein B5R as a fixed product. Gordonii SRL39 strain was transformed with the pLEX plasmid carrying the A27L gene. Since the recipient strain already possessed resistance to erythromycin, it was not possible to screen the pLEX1.1: a271 (a) transformed SRL39 clone using erythromycin selection. Therefore, pLEX1.1: a271 (a) was modified to confer chloramphenicol resistance (Cm ^R ).

PAM401 as template and primer ML16 (5′-GCT AAA AAT TTG TAA TTA AGA AGG AGT GAT TAC CTC GAG ATG ACT TTT AAT ATT ATT GAA TTA GAA AAT TGG-3 ′; SEQ ID NO: 19 ′ The chloramphenicol resistance gene was amplified by PCR using CCA AAT TTA CAA AAG CGA CTC ATA GAA CAT ATG CTA AAT CCA ATC ATC TAC CCT ATG-3 ′, SEQ ID NO: 20). The 3 ′ region of each primer possesses homology with the Cm ^R gene of pAM401, and the 5 ′ sequence is homologous to the region of pLEX1.1: a271 (a) adjacent to the gene conferring erythromycin resistance. is there. The 720 bp product from the PCR reaction was used to initiate a PCR reaction using pLEX1.1: a271 (a) as a template. The product from this reaction was digested with DpnI to remove the parental plasmid and transformed into E. coli XL-10 gold strain. Transformants were screened based on chloramphenicol resistance and sensitivity to erythromycin. Replacement of the erythromycin resistance gene on pLEX1.1: a271 (a) was confirmed by DNA sequencing. This plasmid construct was named pLEX1.3: a271 (a) (SEQ ID NO: 3).

  pLEX1.3: Transformation of SRL39 with a27l (a). S. After confirming B5R expression in the Gordonii SRL39 strain, this strain was transformed with pLEX1.3: a271 (a) to express an additional vaccinia virus antigen A27L. PLEX1.3: a271 (a) was introduced into SRL39 using CSP to facilitate plasmid uptake. Transformants were screened for chloramphenicol resistance. The presence of A27L on the surface of selected clones was assessed using flow cytometry analysis. GP251, SRL21, and newly acquired clone (named SRL46) provided by mouse monoclonal antibody developed against A27L (Dr. Jay Hooper (USAMRIID, Fort Detrick, Maryland)) And labeled for analysis using a FITC-conjugated goat anti-mouse IgG antibody. The fluorescence intensity of 10,000 particles was recorded for each sample using a Beckman Coulter FC500 flow cytometer. Distribution histograms from these analyzes are presented in FIG.

  A substantial shift to a larger fluorescence channel compared to GP251 was observed in both SRL21 and SRL46 samples. This analysis is described in S.H. This suggests that the introduction of pLEX1.3: a271 (a) into the Gordoni B5RΔ expressing strain promoted surface expression of the additional vaccinia virus antigen A27L.

  All cited patents and publications mentioned in this application are hereby fully incorporated by reference for all purposes. The priority application, US Provisional Application No. 60 / 572,974, filed May 21, 2004, is also fully incorporated herein by reference for all purposes.

Schematic of BH4XCRR molecule showing 4 tandem repeat CRRs with a 7 amino acid spacer intervening. The N-terminal 19 amino acids remain after the GTF signal sequence is cleaved during the native processing reaction. Overview of the arrangement of genetic factors in the PLEX system. A 2.3 kbp region was cloned into pVA838 using the BamHI and SphI restriction sites on the plasmid. Putative promoters for rgg and gtfG are identified by right-angled arrows. The shaded region in GTF corresponds to 105 bases encoding a 35 amino acid secretion signal sequence. The heterologous structural gene bh4xcrr is located (functionally) in frame downstream of the secretory signal sequence. pLEX 1.0: S. by bh4xcrr. Gordoni transformation enhances production compared to SPEX. pLEX1.0: bh4xcrr transformation Proteins in culture supernatants from Gordonii and SPEX strain SRL16 were separated using SDS-PAGE, transferred to a PVDF membrane, and probed with monoclonal antibody (mAb) 10F5. We retained the transformant indicated by the asterisk and named this clone SRL44. In dH ₂ O 1: 5 to record the OD ₆₅₀ value of the diluted culture supernatant, blank was subtracted by dH ₂ O. A comparison of the BH4XCRR in the SRL44 culture supernatant to the BH4XCRR standard concentration is presented. Panel A. SDS-PAGE was performed on SRL44 culture supernatant and BH4XCRR diluted to standard concentration. After electrophoresis, the protein was transferred to a PVDF membrane and probed with monoclonal (mAb) 10F5. The BH4XCRR stock solution used as a standard was previously made using SPEX, purified using metal affinity chromatography, and protein using the bicinchoninic acid assay (Pierce (Rockford, IL)). The concentration was determined. Panel B. The band intensity was quantified using concentration measurement, and a regression line was calculated from the value. Band intensities from the SRL44 culture supernatant are plotted on the chart as white diamonds and the BH4XCRR standard is represented by black squares. Primary cleaning and extraction of BH4XCRR from 1 L of SRL44 culture supernatant (SP) is achieved using hydrophobic interaction chromatography. SDS-PAGE was performed using samples of culture supernatant, flow-through (FT), washing solution (W), and elution fraction (3-23). The protein is visualized after staining with Coomassie Brilliant Blue, and BH4XCRR is represented by the major band in these gels. Gel filtration chromatography is used to isolate complete BH4XCRR. The partially purified product (PP) was loaded onto a gel filtration column equilibrated with citric acid. Using a flow rate of 1 mL / min, collection of fractions (5 mL) was started 30 minutes after loading the sample. The elution time (min) of each fraction is shown above each lane. SDS-PAGE was performed on these samples and included a molecular weight marker (M). The major band on these gels, BH4XCRR, was visualized after staining with Coomassie Brilliant Blue. pLEX1.1: a271 (a) transformation Gordonii expresses surface-fixed vaccinia virus antigen A27L. GP251 (negative control), SRL21 (SPEX S. Gordoni A27L; positive control), and SRL45 (PLEX S. Gordoni :: A27L) were grown to OD ₆₅₀ = 0.5. Cells were incubated with anti-vaccinia virus rabbit serum and labeled using FITC-conjugated anti-rabbit IgG. The fluorescence intensity of 10,000 particles was quantified using a Beckman Coulter FC500 flow cytometer. The superimposed distribution histogram is shown. The heterologous gene product B5R is obtained from S. cerevisiae using the SPEX system. It can be expressed on the surface of Gordoni. GP251 (negative control) and SRL39 (SPEX S. Gordoni :: B5R) grown to OD ₆₅₀ = 0.5 were incubated with rabbit antisera raised against vaccinia virus. Reactive rabbit antibodies were detected using FITC-conjugated anti-rabbit IgG. Flow cytometry was used to record the level of fluorescence associated with 10,000 particles from each culture. A representative distribution histogram is shown. pLEX1.3: B5R expression by a27l (a) Gordoni transformation promotes surface expression of viral antigen A27L. GP251 (negative control), SRL21 (S. gordonii :: A27L positive control), and SRL46 (S. gordonii :: B5R _SPEX A27L _PLEX ) were incubated with a mouse monoclonal antibody developed against A27L to produce a FITC conjugate. Labeled for analysis using goat anti-mouse IgG antibody. The fluorescence intensity of 10,000 particles was recorded for each sample using a Beckman Coulter FC500 flow cytometer. A superimposed representative distribution histogram from these analyzes is presented. S. Illustration of BH4XCRR sequence within Gordoni. The BH4XCRR nucleic acid sequence (SEQ ID NO: 21) and the corresponding amino acid sequence (SEQ ID NO: 22 and 23) insert the structural gene sequence in frame downstream of the positive regulator rgg and the gtfG promoter and signal sequence. Obtained by Putative promoters are shown at positions -35 and -10. The Shine-Dalgarno ribosome binding site is labeled “SD”. The depicted sequence was then inserted into the E. coli / Streptococcus shuttle plasmid pVA838 and then competent S. Gordonii cells were transformed. Alternative shuttle plasmids can also be used. S. Illustration of BH4XCRR sequence within Gordoni. The BH4XCRR nucleic acid sequence (SEQ ID NO: 21) and the corresponding amino acid sequence (SEQ ID NO: 22 and 23) insert the structural gene sequence in frame downstream of the positive regulator rgg and the gtfG promoter and signal sequence. Obtained by Putative promoters are shown at positions -35 and -10. The Shine-Dalgarno ribosome binding site is labeled “SD”. The depicted sequence was then inserted into the E. coli / Streptococcus shuttle plasmid pVA838 and then competent S. Gordonii cells were transformed. Alternative shuttle plasmids can also be used.

Claims (23)

  A nucleic acid comprising an operably linked rgg gene, a gtfG promoter, and a nucleic acid encoding a polypeptide of interest, wherein the polypeptide of interest is a fusion protein of a protein and a GTF signal sequence.   The nucleic acid of claim 1, wherein the rgg gene, the gtfG promoter, and / or the GTF signal sequence is derived from Streptococcus.   The nucleic acid of claim 2, wherein the rgg gene, the gtfG promoter, and the GTF signal sequence are derived from Streptococcus.   Streptococcus is S. The nucleic acid of claim 2 which is gordonii.   2. The nucleic acid of claim 1, wherein the polypeptide of interest comprises an antigen, antibody or fragment thereof, immunogenic polypeptide, pharmaceutical agent, or enzyme.   The nucleic acid encoding the polypeptide of interest further comprises a sequence encoding an LPXTG (X can be any amino acid) (SEQ ID NO: 24) anchoring motif at the carboxy terminus of the polypeptide of interest. Nucleic acids.   2. The nucleic acid of claim 1, wherein the polypeptide of interest is BH4XCRR.   2. The nucleic acid of claim 1, wherein the nucleic acid encoding the polypeptide of interest is operably linked to the nucleic acid encoding the purified polypeptide, and the purified polypeptide contains a cleavage site.   A vector comprising the nucleic acid of claim 1, wherein the nucleic acid is functionally inserted into the vector.   10. The vector of claim 9, comprising an Escherichia and / or Streptococcus replicon.   The vector of claim 9 comprising a polylinker and / or an antibiotic selection gene.   A host cell transformed with the vector of claim 9.   13. The host cell of claim 12, further comprising a surface protein expression (SPEX) vector.   The host cell according to claim 12, which is a Streptococcus bacterium, an Escherichia bacterium, a Lactococcus bacterium, or a Lactobacillus bacterium.   Streptococcus sp. 15. The host cell of claim 14, which is a Gordoni.   A polypeptide of interest synthesized from the host cell of claim 12.   A polypeptide of interest synthesized from the host cell of claim 12 which is substantially purified.   14. A polypeptide of interest synthesized from the host cell of claim 13 which is substantially purified.   16. A polypeptide of interest synthesized from the host cell of claim 15 that is substantially purified. A method for synthesizing a heterologous polypeptide of interest in a host cell comprising:
(A) functionally linking the nucleic acid of claim 1 to a vector;
(B) inserting the vector into a host cell;
(C) culturing host cells that express the nucleic acid under conditions under which the polypeptide of interest is synthesized; and (d) a method comprising isolating and purifying the polypeptide of interest.   21. The method of claim 20, wherein the host cell is Streptococcus or Escherichia.   The host cell is S. cerevisiae. The method of claim 21, which is a Gordoni.   21. The method of claim 20, further comprising the step of cotransforming the host cell with a surface protein expression (SPEX) vector comprising a second polypeptide of interest.

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