EP1765854A1

EP1765854A1 - An expression system comprising operably linked rgg gene and gtfg promoter

Info

Publication number: EP1765854A1
Application number: EP05754796A
Authority: EP
Inventors: Travis K. Warren; Kevin F. Jones; Dennis E. Hruby
Original assignee: Siga Technologies Inc
Current assignee: Siga Technologies Inc
Priority date: 2004-05-21
Filing date: 2005-05-23
Publication date: 2007-03-28
Also published as: JP2008501364A; AU2005245982A1; EP1765854A4; CA2567501A1; WO2005113583A1

Abstract

Provided is a plasmid-based expression system that can be used to enhance production of C-repeat region (CRR) by Streptococcus gordonii, other Strpetococcus strains, as well as other bacteria. This system utilizes the gtfG promoter and secretion-signal sequence in combination with the positive regulatory gene, rgg, to drive the synthesis and secretion of CRR from the resident Escherichia. coli or Streptococcus shuttle plasmid, pVA838. This plasmid isolated from E. coli was readily introduced into S. gordonii and under optimal conditions led to a nearly 10-fold increase in the concentration of secreted protein relative to production by surface protein expression system (SPEX).

Description

A Plasmid-Borne Expression (PLEX) System for the Production of Heterologous Gene Products by the Gram-positive Bacterium, Streptococcus gordonii

REFERENCE TO A SEQUENCE LISTING The attached sequence listing is incorporated herein by reference in its entirety.

BACKGROUND Escherichia coli-based expression systems are widely used for the production of heterologous protein products. However, these expression systems possess limited usefulness for production of particular proteins that are sensitive to degradation by E. coli proteolytic enzymes, expressed in inclusion bodies, or are produced at unacceptably low levels. Thus, a need remains to produce recombinant proteins that are readily obtained in high quantities that maintain the protein's natural biological activity.

SUMMARY Provided herein, among other aspects, is a novel system of expression in which a resident plasmid is employed to enhance expression of heterologous proteins and polypeptides. In one example, the heterologous antigen BH4XCRR is expressed in S. gordonii. Using this system, the regulatory gene rgg, for example, is employed to direct expression of BH4XCRR while secretion is facilitated by fusion of the BH4XCRR structural gene to the GTF secretion-signal sequence. Thus, the product of the rgg gene (the regulatory gene) in turn regulates GTF, the regulated gene. Also provided is a chemically defined medium that permits production of the protein and that facilitates its purification. The PLEX system has proven useful for expression of an intact protein product, BH4XCRR, that was 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 not amenable to expression by E. coli. Because PLEX is compatible with the SPEX expression system, both systems may be employed within a single clone to express either individual or multiple proteins. PLEX also serves as a useful tool for the expression of heterologous antigens on the surface of Streptococcus gordonii, which then may be used as, for example, a vaccine vector. Other bacterial species and strains can also be used. Provided herein are nucleic acids comprising an operably linked rgg gene, a gtfG promoter, and a nucleic acid which encodes a polypeptide of interest; the polypeptide of interest when synthesized is a fusion protein of a polypeptide of interest and a GTF 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, the gtfG promoter, and/or the GTF signal sequence preferably are from Streptococcus, but can be from another bacterial strain. Preferred bacteria are S. gordonii. The polypeptide of interest can include but is not limited to an antigen, an antibody, an immunogenic fragment of an antibody, an enzyme, or any other protein discussed herein as well as small peptides and pharmaceutical agents. A preferred polypeptide of interest is that denoted by BH4XCRR and other polypeptides that may be used immunogenic pharmaceutical compositions (e.g., vaccines). Preferably the regulatory gene (rgg gene) is from the same organism as the GTF signal sequence. For example, both the rgg gene and the GTF signal sequence would come from S. gordonii or from another Streptococcus. However it is also contemplated that the regulatory gene, the rgg gene of one species could be matched, for example, to a closely related GTF signal sequence of another species of bacteria. If used in an immunogenic pharmaceutical composition, preferably the composition contains pharmaceutically acceptable carriers, excipients, and the like. If in the form of a pharmaceutical composition for the purpose of raising an immune response, the composition may further comprise adjuvants. The portion of the nucleic acid encoding the polypeptide of interest may further comprise a second sequence encoding a purification polypeptide. Examples include but are not limited to proteins that assist with purification such as glutathione-s-transferase (GST) and hemagglutanin (HA). Preferably the purification polypeptide comprises a cleavable domain such that the protein of interest can be cleaved from the purification polypeptide. Other sequences that may be used to facilitate purification and/or detection include but are not limited to 6X-histidine tags and Flag tags, which would be fused to the carboxy terminus of the BH4XCRR sequence. Another aspect has the nucleic acid of interest operably incorporated in a vector. These vectors can further comprise suitable replicons, such as Escherichia and/or Streptococcus replicons. Preferable replicons can be those that provide a higher plasmid copy number to enhance the quantity of recoverable DNA from, for example, £. coli. By increasing plasmid copy number, the amount of heterologous products are correspondingly increased as a result of maximizing the number of production-related genes in each cell. The vectors may further comprise one or more polylinkers and/or antibiotic selection genes. Preferred polylinkers would be inserted as between the Kpn\ and EcoRI restriction recites to facilitate cloning of heterologous genes and/or fusion genes into the plasmid that may have one or more Kpn\ and/or EcoRI restriction sites. The vectors are capable of replication and polypeptide expression in a host cell. Suitable antibiotic genes may include chlroamphenicol (erythromycin if not already present), kanamycins (e.g., neomycin), tetracycline (tet^R), ampicillin (Amp^R), and/or carbenicillin. Another aspect of the invention has the vectors capable of expression transformed or transfected in a host cell. Preferred organisms for transformation include Escherichia, Streptococcus, Lactococcus, and Lactobacillus bacteria, especially E. coli, S. gordonii, L lactis, and Lactobacillus sp. In yet a further embodiment, the host cells can be co-transformed or co-transfected with a surface protein expression (SPEX) vector. By co-transformed/co-transfected is meant a host cell wherein the host cell has vectors including the SPEX and PLEX vectors inserted at the same time, or wherein one vector is inserted first followed by transfection with the second vector. Another aspect contemplates the purification of the polypeptide of interest.

Polypeptide can be purified from the supernatant of the cultured host cells. Alternatively, if the polypeptide of interest expresses the LPXTG (SEQ ID NO: 24) anchoring motif, the polypeptide of interest may be collected in combination with the cell wall for use as an antigen. Preferably the polypeptide of interest is a substantially purified polypeptide. By substantially purified is meant that when the protein is resolved via PAGE, only the protein of interest is observed after Coomassie staining of the gel. Yet another aspect contemplates a method of synthesizing a heterologous polypeptide of interest in a host cell comprising: (a) operably linking the nucleic acid of claim 1 to a vector, wherein said nucleic acid encodes a polypeptide of interest; (b) inserting the vector in the host cell; (c) culturing the host cell expressing the nucleic acid under conditions wherein the polypeptide of interest is synthesized; and (d) isolating and purifying the polypeptide of interest. The method can use any host cell, but preferably the host cell is a

Streptococcus or an Escherichia, a Streptococcus, a Lactococcus, and a Lactobacillus host cell. The method can further comprise a step of co-transforming or co-transfecting the host cell with a surface protein expression (SPEX) vector comprising a second polypeptide of interest.

BRIEF DESCRIPTION OF THE DRAWINGS The drawings are exemplary only, and should not be construed as limiting the invention. FIG. 1. Representation of the BH4XCRR molecule showing four tandemly- repeated CRRs with intervening 7-amino acid spacers. The 19 amino acids at the N-terminus remain after the GTF signal sequence is cleaved during native processing reactions. FIG. 2. Overview of the arrangement of genetic elements in the PLEX system. A 2.3 kbp region was cloned into pVA838 using the BamHI and Sphl restriction sites on the plasmid. The putative promoters for rgg and gffG are identified by right-angled arrows. The shaded region in GTF corresponds to the 105 bases coding for the 35-amino acid secretion signal sequence. The heterologous structural gene, bh4xcrr, is situated downstream and (operably) in-frame with the secretional signal sequence. FIG. 3. Transformation of S. gordonii with pLEX1.0:btj4xcrr enhances production relative to SPEX. Protein in culture supematants from pLEX1.0:bh4xcrr -transformed S. gordonii and from SPEX strain SRL16 were separated using SDS- PAGE, transferred to PVDF membrane and probed with monoclonal antibody (mAb) 10F5. We retained the transformant indicated with the asterisk, and designated this clone as SRL44. The OD₆₅₀ values were recorded in culture supematants diluted 1 :5 in dH₂O and blanked against dH₂O. FIG. 4. Comparison of BH4XCRR in SRL44 culture supematants with BH4XCRR standard concentrations is presented. Panel A. SDS-PAGE was conducted on SRL44 culture supernatant and BH4XCRR diluted to standard concentrations. After electrophoresis, proteins were transferred to PVDF membrane and probed with monoclonal (mAb) 10F5. The stock solution of

BH4XCRR used for standards had been previously produced using SPEX, purified using metal-affinity chromatography, and the protein concentration determined using a bicinchoninic acid assay (Pierce, Rockford, IL). Panel B. Band intensities were quantified using densitometry, and a regression line was calculated from the values. The band intensity from SRL44 culture supernatant is plotted on the chart as an open diamond, and the BH4XCRR standards are represented by closed squares. FIG. 5. Initial cleanup and extraction of BH4XCRR from 1 L SRL44 culture supernatant (SP) is accomplished using hydrophobic-interaction chromatography. SDS-PAGE was conducted using a sample of culture supernatant, flow through (FT), wash (W) and eluted fractions (3-23). Proteins were visualized after staining with Coomassie Brilliant Blue, and BH4XCRR is represented by the predominant bands in these gels. FIG. 6. Gel filtration chromatography is used to isolate intact BH4XCRR. A partially purified product (PP) was loaded onto a citric-acid equilibrated gel-filtration column. Using a flow rate of 1 mL/min, fractions (5 mL) were collected beginning 30 min after loading the sample. Elution times (min) for each fraction are indicated above each lane. SDS-PAGE was conducted on these samples and included a molecular weight marker (M). BH4XCRR, the predominant band on these gels, was visualized after staining with Coomassie Brilliant Blue. FIG. 7. pLEX1.1 :a27l(a) transformed S. gordonii express surface-anchored vaccinia viral antigen A27L. GP251 (negative control), SRL21 (SPEX S. gordonii A27L; positive control), and SRL45 (PLEX S. gordonii:Λ27L) were grown to an OD₆₅o=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. Overlaid distributional histograms are shown. FIG. 8. Using the SPEX system, heterologous gene product B5R can be expressed on the surface S. gordonii. GP251 (negative control) and SRL39 (SPEX S. gordonii::B5R), grown to an OD₆so = 0.5, were incubated with rabbit anti-serum raised against vaccinia virus. Reactive rabbit antibodies were detected using FITC- conjugated anti-rabbit IgG. Using flow cytometry, the level of fluorescence associated with 10,000 particles from each culture was recorded. Overlaid representative distributional histograms are shown. FIG. 9. Transformation of B5R-expressing S. gordonii with pLEX1.3:a27l(a) facilitates the surface expression of viral antigen A27L. GP251 (negative control), SRL21 (S. gordonii: .A27L positive control), and SRL46 (S. goΛ /on//.: B5R_Sp_EχA27Lp_LEχ) were incubated with a mouse monoclonal antibody developed against A27L and labeled for analysis using 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. Overlaid representative distributional histograms from these analyses are presented. FIG. 10. Depiction of BH4XCRR sequence in S. gordonii. The BH4XCRR nucleic acid sequence (SEQ ID NO: 21 ) and corresponding amino acid sequences (SEQ ID NO: 22 and 23) were obtained by insertion of the structural gene sequence in-frame and downstream of the positive regulator, rgg, and the gtfG promoter and signal sequence. Putative promoters are indicated at their -35 and -10 sites. Shine-Delgamo ribosomal binding sites are denoted by "SD". The depicted sequence was then inserted into the E. coli/Streptococcus shuttle plasmid pVA838, which was then transformed in to competent S. gordonii cells. Alternative shuttle plasmids can also be used.

DETAILED DESCRIPTION The PLasmid-borne Expression (PLEX) system is a technique useful for the production of heterologous protein products. This is accomplished by the insertion of specific endogenous and heterologous genes and nucleic acids expressing polypeptides of interest in to a suitable plasmid vector; the vector is then transformed into the Gram-positive bacterium S. gordonii. Both the specific inserted sequences that facilitate production and their placement on a resident plasmid distinguish this invention as unique from previously established expression systems. During growth using typical in vitro culture conditions, S. gordonii secretes the 172 kDa glucosyltransferase (GTF) in to the culture supernatant. A positive regulatory gene, rgg, has been identified in S. gordonii. When rgg is over- expressed using plasmid complementation methods, the rgg gene enhances GTF activity in culture supematants. Three genetic elements -- rgg, portions of gtfG, and the heterologous gene sequence, are required for production and secretion of the heterologous gene product of interest. Expression of the heterologous gene product is driven by the endogenous promoter for gtfG. Expression from the gtfG promoter is positively regulated by Rgg. Secretion of the product is facilitated by fusion of the heterologous product to the GTF signal sequence. These genetic elements have been inserted for the purposes of this invention into an appropriate plasmid. The plasmid chosen for this role is pVA838, which is publicly available through the American Type Culture Collection (ATCC). Characteristics of this plasmid that make it suitable for this application include cloning sites for insertion of genetic material, the presence of antibiotic resistance markers for the detection of transformants, and the presence of both E. coli and Streptococcus replicons. Alternative plasmid vectors can be utilized. Also, additional genetic elements can be inserted into the vector or in the cassette comprising the above-referenced three genetic elements, including but not limited to elements with additional cloning sites (polylinkers), cassettes for color selection, antibiotic resistance genes (e.g., ampicillin, chloramphenicol), different polymerase promoters (e.g., T7 and T4), and nucleic acids for additional fusion protein sequences to allow for rapid column purification and thereafter cleavage of the protein used for purification. The PLEX system can be easily utilized with standard molecular biology techniques and traditional microbiological laboratory equipment and reagents. The PLEX system possesses an enhanced level of expression of model protein, BH4XCRR, relative to the most comparable expression system known, the Surface Protein Expression (SPEX) system. The SPEX system is described in detail in U.S. Patent No. 5,821 ,088, the contents of which are incorporated herein by reference in its entirety for all purposes. Relative to SPEX, it was estimated that the PLEX expression system secreted nearly ten-fold the amount of model product BH4XCRR into the culture supernatant when bacteria were cultured using previously identified optimal growth conditions. The concentration of BH4XCRR in the culture supernatant was estimated at 10 mg/L using the PLEX system by comparison with previously purified BH4XCRR standard concentrations. The PLEX system additionally overcomes certain limitations associated with prior E. coli expression systems. Prior E. coli expression systems are limited in their ability to express particular gene products, including products that are expressed in inclusion bodies, products that serve as substrates for E. coli proteolytic enzymes, or those that otherwise fail to exhibit a high level of expression by that bacterium. Additionally, concerns arise when an E. co//-expressed protein product is to be used in an immunological capacity, as small amounts of endotoxin, unintentionally co-purified along with the protein product of interest, can complicate studies. Use of the PLEX system eliminates these concerns because proteins are produced by a Gram-positive bacterium, which do not contain endotoxin. Applications of the PLEX system can be easily expanded to include the expression of antigens using a bacterial commensal vector such as S. gordonii. A commensal vector such as S. gordonii can be used to generate an immunological response to surface-expressed heterologous antigens. Using PLEX, heterologous proteins can be anchored to the surface of a commensal vector such as S. gordonii, instead of secreted, by addition of an LPXTG (SEQ ID NO: 24) anchoring motif to the C-terminus of the heterologous product. Additionally, multiple antigens can be expressed, either as secreted molecules or anchored, by transforming a commensal vector such as S. gordonii with multiple PLEX-system plasmids. Also described herein are materials and methods for using E. coli expression vectors to produce a streptococcal subunit-vaccine candidate, designated BH4XCRR. BH4XCRR is a circa 45 kDa antigen comprised of four C- repeat regions from the Streptococcus pyogenes M6 protein (FIG. 1 ). These efforts have shown that BH4XCRR is susceptible to degradation when expressed in E. coli. However, it can be expressed and secreted in an intact form by Gram-positive S. gordonii using the Surface Protein Expression (SPEX) System. Using SPEX, a heterologous structural gene, such as bh4xcrr, is cloned in to (operably linked) plasmid pSMB104 so that the structural gene is flanked by regions homologous to the S. gordonii chromosome and regions corresponding to the emmβ.1 gene from S. pyogenes (Myscofski et al., 1998 Protein Expression and Purification 14: 409-417). The homologous flanking regions facilitate chromosomal integration of the structural gene downstream of a P2 promoter, while fusion of the heterologous gene to 16 amino acids (aa) of the N-terminal region of the S. pyogenes M6 protein provides the secretion-signal sequence. The SPEX system has been successfully used for the production of numerous heterologous proteins including derivatives of S. pyogenes 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 staphylococcal nuclease A (Dutton et al., 2000 Protein Expression & Purification 19: 158-172). However, yields of BH4XCRR from the SPEX system have been insufficient for the intended applications, for example due to low expression of the protein and inefficient recovery methods for the protein. During growth using typical in vitro culture conditions, i.e. using Todd Hewitt broth with yeast extract (THY) or Brain Heart Infusion (BHI) broth, S. gordonii secretes the 172 kDa enzyme glucosyltransferase (GTF) into the culture supernatant. This enzyme synthesizes glucan polymers from sucrose, which may play an important role in the in vivo formation of a microbial biofilm 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). Examinations into the regulatory mechanisms suggest that Rgg may act to enhance both transcription and translation of gtfG (Vickerman et al., 1997 Applied & Environmental Microbiol. 63: 1667-1673). Structural predictions of Rgg suggest the presence of a helix-turn- helix motif indicative of a DNA-binding protein (Bateman et al., 2000 Nuc. Acids Res. 28: 399-406). Interactions between Rgg and sequences located in the 3' region of rgg may facilitate transcriptional activity at the GTF promoter (Sulavik et al., 1992 J. Bacteriol. 174: 3577-3586; and Vickerman et al., 2003 Microbiology 149: 399-406). Rgg may be capable of enhancing translation of GTF by melting a predicted hairpin-loop secondary structure that sequesters the gtfG ribosomal- binding site on polycistronic rgg/gtfG mRNA (Sulavik et al., 1992 J. Bacteriol. 174: 3577-3586). When over expressed in S. gordonii using plasmid complementation, Rgg enhanced the levels of GTF activity in culture supematants by approximately six-fold (Sulavik et al., 1992). The system of PLEX uses the interaction of Rgg to enhance activity at the gtfG promoter as the means of expressing heterologous polypeptides of interest. PLEX can be used to produce either secreted or surface-anchored heterologous products by S. gordonii. Using this system, the regulatory gene rgg is incorporated into the plasmid to direct expression of a structural gene of interest from the gtfG promoter. Secretion of the product is facilitated by fusion of the structural gene with the GTF secretion-signal sequence. Also described is a combination of unique culture conditions that facilitate rapid and efficient purification of the secreted product. Expression of heterologous products on the surface of S. gordonii is accomplished by fusing the heterologous product with anchoring sequences derived from the S. pyogenes M protein. Additionally, described herein are uses of the PLEX system for the expression of multiple heterologous antigens on the surface of S. gordonii. The expression system described herein can be used to express any polypeptide of interest from a pathogen, including enzymes, antigens for use in preparing immunogenic preparations, signaling proteins, and the like. Preferably, the expression is in a Streptococcus strain, especially in S. gordonii. Preferred proteins of interest are proteins that are secreted or expressed on the surface of the organism from which they are derived. The expression system can also be utilized to express proteins directly into the culture media for ready collection of the recombinantly produced protein from the culture media. Proteins of interest can include antigens, enzymes, surface- expressed proteins, eukaryote-derived proteins, polypeptide fragments, pharmaceuticals, antibodies (fragments of antibodies such as scFv, Fab, F(ab')₂ and the like), and immunogenic polypeptides. 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 wherein 80% or more of the polypeptide or nucleic acid are the same. For example, a substantially purified polypeptide would not have other polypeptide contaminants synthesized from another nucleic acid. Acronyms and definitions as provided herein are art accepted unless indicated otherwise. It must be noted that 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 reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth. Acronyms

Ab antibody

BHI brain heart infusion broth

CAA casamino acids

CDM chemically defined medium Cm^R chloramphenicol resistance

CRR C-repeat region

CSP competence stimulating peptide(s)

CV column volumes dH₂O distilled water ELISA enzyme-linked immunosorbent assay

GST glutathione-s-transferase

GTF glucosyltransferase

HA hemagglutanin

IFN-γ interferon gamma IgG immunoglobulin G (gamma)

IL-2 interleukin-2 kbp kilobase pair kDa kilo Dalton

LB Luria-Bertani broth mAb monoclonal antibody

PCR polymerase chain reaction

PLEX plasmid-borne expression system

PVDF polyvinylidene fluoride (membrane)

SDS-PAGE sodium dodecylsulphate polyacrylamide gel electrophoresis SPEX surface protein expression system

THY Todd Hewitt broth with yeast extract EXAMPLES Although the present invention has been described in detail with reference to the examples below, it is understood that various modifications can be made without departing from the spirit of compositions and methods disclosed herein.

EXAMPLE 1 Secretion of heterologous products The functionality of the PLEX system was tested by expressing an approximately 45 kDa protein product (denoted BH4XCRR), which is modeled on the M-protein C-repeat region (CRR) from S. pyogenes. FIG. 2 details the placement and alignment of rgg, gtfG, and bh4xcrr coding sequences as they were positioned on the plasmid to facilitate production of the BH4XCRR protein. Bacterial Strains and Growth Conditions. E. coli strains were grown in Luria-Bertani (LB) broth (Difco, Detroit, Ml) or on LB agar (Difco). S. gordonii was grown in Brain-Heart Infusion (BHI) (Difco) or on BHI containing 1.5% agar. For protein production, S. gordonii was cultured in chemically defined medium (CDM) (JRH Biosciences, Lenexa, KS), containing 5% casamino acids (CAA) (Fisher Scientific, Pittsburgh, PA). Selection and growth for strains carrying antibiotic- resistance determinants were performed at 25 μg chloramphenicol/mL for E. coli strains and at 5 μg erythromycin/mL for S. gordonii. DNA Isolation and Manipulation. Standard molecular biological techniques were followed in the preparation and manipulation of DNA. PCR products were cloned into pCR2.1 TOPO (Invitrogen, Carlsbad CA) and transformed into E. coli InvαF' (Invitrogen) using the manufacturer's procedure. Plasmids were isolated from E. coli strains using Qiagen purification kits (Valencia, CA). Following digestion with endonucleases, DNA fragments were separated by electrophoresis and eluted from agarose gels using 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). When transformed into E. coli, pVA838 confers resistance to chloramphenicol and erythromycin. In S. gordonii, pVA838 only provides erythromycin resistance. Amplification of rgg/gtfG. Primers (forward primer TW3 5'- CCGGGATCCCGTTGACGGAGATTAG-3', SEQ ID NO: 4, and reverse primer TW4 5'-CCGGGTACCCCTGAACAGCGGACTGTTC -3', SEQ ID NO: 5) were designed for the amplification of a 1.3 kbp product using S. gordonii Challis chromosome as template. These primers were designed based on the published sequence of rgg and the 5' region of gtfG (GenBank Accession No.: M89776). TW3 introduced a BamHI restriction site at the 5' end of the rgg sequence. PCR using TW3 and the reverse primer generated a Kpnl restriction site at the 3'-end of the gtfG sequence. Design and Amplification of BH4XCRR. BH4XCRR contains four identical domains that are modeled on the conserved C-repeat region (CRR) of the M protein from S. pyogenes (FIG. 1 ). Each CRR segment contains 70 amino acids. At the N- terminus of the molecule and between each CRR segment are 7-amino acid spacers, each of which has a different amino acid sequence, but has the proper sequence content to maintain the alpha-helical coiled-coil nature of the molecule (Phillips et al., 1981 Proc. Nat'l. Acad. Sci. USA 78: 4689-4693). A six-residue histidine tag was included at the C-terminus with the original intent of expanding purification options. In addition, the nucleotide sequence for each CRR segment was altered as much as possible, while keeping the amino-acid sequences of each segment identical. Last, the codon usage has been optimized for expression in S. gordonii. The gene was synthesized by Blue Heron Biotechnology (Bothell, WA) and was supplied as an insert in a purified plasmid. Using this plasmid as template, PCR was performed with forward primer TW5 (5'-

CCCGGTACCTGAAACAAGAGTTAGACGAGGG-3', SEQ ID NO: 6) and reverse primer TW6 (5'-ACATGCATGCTTAATGATGGTGATGATGGTGTTG-3\ SEQ ID NO: 7) to amplify the bh4xcrr structural gene. Amplification of bh4xcrr was conducted to introduce a Kpnl restriction site on the 5' end along with two additional nucleotides to facilitate in-frame fusion with the gtfG secretion-signal sequence. PCR amplification was also used to introduce a Sphl restriction site and a stop codon on the 3' end of bh4xcrr. Construction of pLEX1.0:bh4xcrr. For cloning, pCR2.1 containing the rgg/gtfG insert was sequentially digested, first with Kpnl and then with BamHI. Digestion with Kpnl was conducted using the manufacturer's recommended reagents for 5 h at 37^°C. Sodium chloride was adjusted to 50 mM and BamHI was added to the reaction for an additional 5 h. DNA fragments were separated using agarose-gel electrophoresis and a 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 a simultaneous double digestion with Sphl and Kpnl, according to the manufacturer's suggested protocol, for 3 h at 37^°C. A 1 kbp DNA fragment was resolved using electrophoresis. This band was excised, extracted from the agarose matrix, and stored at -20^°C for future use. Plasmid pVA838 was simultaneously digested using BamHI and Sphl restriction enzymes according to the manufacturer's recommended reaction conditions for 4 h at 37^°C. A circa 10 kbp DNA fragment was excised from the gel following electrophoresis, was extracted and stored at -20^°C. The bh4xcrranά rgg/gtfG fragments were cloned into BamHI- and Sphl- digested pVA838 during a triple-ligation reaction. Digested DNA was added to the reaction at a quantity ratio of 5:5:1 Ligation was accomplished during an overnight incubation at 13^°C with T4 DNA ligase (Promega, Madison, Wl), using the manufacturer's suggested reaction conditions. The ligation product was immediately transformed into the InvαF' strain of E. coli. Transformants bearing pVA838:rgg/gtfG/bh4xcrr were selected from chloramphenicol-resistant colonies. Further selection was based on isolation of an appropriately sized 11.5 kb plasmid, and by PCR using isolated plasmid as template and primers TW3 and TW6. The DNA sequence of the insert was confirmed using sequencing primers TW7 (5'-GCAACAGAAATATCACCTGCCG-3'; SEQ ID NO: 8), TW8 (5'-CTTGTGGCTCCAAAGGCCTTG-3'; SEQ ID NO: 9), and TW9 (5'-GGACCTTGCCAACTTGACCGC-3'; SEQ ID NO: 10). The plasmid construct was designated pLEXI .Q:bh4xcrr (see FIG. 2; SEQ ID NO: 1 ). Purified pLEX1.0:bMxc/τwas introduced into S. gordonii GP251 using previously established transformation methods (Pozzi et al., 1990 Res. Microbiol. 141 : 659-670). Briefly, S. gordonii were grown in BHI in the presence of 10% fetal calf serum to induce competence and stored at -80^°C in 450-μl aliquots. Plasmid pLEXI .0:bh4xcrr was incubated with freshly thawed competent cells for 2 h at 37^°C. This culture was mixed with liquified BHI agar containing 5% defibrinated sheep blood and layered onto BHI agar. After briefly allowing this layer to congeal, liquified BHI agar containing 5 μg erythromycin/mL was carefully poured onto the plates. Colonies, selected after 48 h growth at 37^°C, were streaked onto BHI agar containing 5 μg erythromycin/mL to further confirm resistance and for selection of pure clones. Transformants were confirmed based on detection using Western blot of BH4XCRR in culture supematants after overnight growth in BHI containing 5 μg erythromycin/mL at 37^°C. Electrophoresis and Western Blot. Proteins in S. gordonii culture supematants were separated using SDS-PAGE. Gels were either stained using Coomassie Brilliant Blue or were transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 3% bovine serum albumin (BSA). Antibodies were diluted in buffer containing 0.5 % Tween 20, 0.02 % sodium azide, 0.5 M sodium chloride, and 0.01 M Tris at pH 8.2. Membranes were probed using mAb 10F5, which is specific for an epitope found in the M-protein C-repeat region of S. pyogenes (Jones et al, 1986 J. Exp. Med. 164: 1226-1238), at a concentration of 2 μg/mL and goat anti-mouse IgG AP-conjugated antibody (BioRad, Hercules, CA) at a dilution of 1 :1000. Blots were developed using AP-conjugate substrate kit (BioRad) diluted in buffer containing 0.06 M Tris, 0.02% sodium azide, 0.06% magnesium chloride hexahydrate at pH 9.8. Following transformation of S. gordonii, erythromycin-resistant colonies were grown overnight at 37^°C in BHI broth. All cultures were back-diluted in CDM/CAA containing erythromycin and the optical density of each culture was monitored until it reached stationary phase (OD₆₅o=0.45 following 1 :5 dilution in dH₂O). Culture supematants were prepared for SDS-PAGE, and BH4XCRR was visualized by Western blot (FIG. 3). Samples were cultured and processed from a BH4XCRR-secreting SPEX strain to permit comparison of production levels between these two expression systems. Intact BH4XCRR was detected in the culture supernatant of eight out of ten pLEXI .0:bA)4xcrr-transformed S. gordonii, and production in all transformants was enhanced relative to production in the SPEX strain (FIG. 3). A single pLEX1.0:b xc/τ-transformed S. gordonii culture was retained and designated as SRL44. While BH4XCRR could be readily detected in culture supematants using Western blots probed with mAb 10F5, numerous approaches to quantify the BH4XCRR concentration in culture supematants using enzyme-linked immunosorbent assay (ELISA) were unsuccessful. As an alternative, using Western blot the BH4XCRR band intensity in SRL44 culture supernatant was compared against those 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 medium at a concentration of 10 mg/L by SRL44. EXAMPLE 2 Production and Purification of Heterologous Product BH4XCRR A frozen stock (-80^°C) of SRL44 was used to inoculate 10 mL BHI containing 5 μg erythromycin/mL, and this culture was incubated at 37^°C overnight. It was back-diluted in 1 L filter-sterilized CDM supplemented with 5% CAA and 5 μg erythromycin/mL and was grown to OD₆so = 0.45 (1 :5 dilution in dH₂O; blanked against dH₂O) in a stationary 37^°C incubator. Cells were pelleted by centrifugation, and the supernatant was filtered using a filter unit with a cutoff of 0.45 μm. In preparation for hydrophobic-interaction chromatography, (NH )₂SO₄ was added to the supernatant at a concentration of 1.5 M, and the supernatant was filtered again to remove precipitates. The supernatant was loaded onto a 5-mL HiTrap Butyl FF column (Amersham BioSciences, Piscataway, NJ) that had been stripped with 5 column volumes (CV) of 20 mM Na₂HPO₄, pH 7.0 and equilibrated with 5 CV of 50 iriM Na₂HPO₄/1.5 M (NH₄)₂SO pH 7.0. After the sample had been loaded, the column was washed with 10 CV of 50 mM Na₂HPO₄/1.5 M (NH₄)₂SO₄ pH 7.0. Proteins were eluted using a 10-CV (NH₄)₂SO gradient from 1.5 to 0 M, and 2-mL fractions were collected. All steps were conducted at a flow rate of 5 mL/min. The effectiveness of this procedure was evaluated by running fractions on an SDS- PAGE gel, and proteins were visualized by staining with Coomassie Brilliant Blue (FIG. 5). BH4XCRR was not detected in a sample of the flow-through taken as the sample was nearly completely loaded onto the column, suggesting that the product was efficiently extracted from the supernatant. BH4XCRR was eluted from the column in a limited number of fractions, and is represented as a single darkly stained band at 45 kDa, suggesting that it remains predominantly intact during this processing step. Additionally, BH4XCRR was separated from a number of other proteins secreted by S. gordonii during gradient elution. Following this initial cleanup, BH4XCRR was further purified using gel- filtration chromatography. Fractions from the hydrophobic-interaction separation that contained BH4XCRR were pooled and proteins were precipitated using a 60% saturated solution of (NH₄)₂SO₄. The precipitate was pelleted, redissolved in 50 mM citric acid pH 2.8, and loaded onto an equilibrated HiPrep16/60 Sephacryl S- 300 column (Amersham). While BH4XCRR was visualized on Coomassie-stained SDS-PAGE gels in fractions that elute 40 to 75 min after sample was loaded, it was predominantly eluted in four 5-mL fractions collected between 50 and 65 min (FIG. 6). These fractions contained undetectable amounts of contaminating proteins. A faint banding pattern was observed below the primary BH4XCRR band in fractions eluted at 60 and 65 min. This banding pattern is indicative of minor BH4XCRR degradation, and the reactivity of these bands has been confirmed using Western blots probed with mAb 10F5. Methods used for purification of BH4XCRR from 1 L SRL44 culture supernatant are provided in Table 1 below. The steps occur in the order in which they are displayed. Table 1

EXAMPLE 3 Expression of anchored heterologous products using the PLEX plasmid Using the genetics associated with the SPEX system heterologous gene products can be anchored to the cell-wall of S. gordonii by fusing a structural heterologous gene with the C-terminal region from the S. pyogenes M-protein, which contains a Gram-positive anchoring motif (Myscofski et al., 1998 Protein Expression & Purification 14: 409-17). By incorporating this anchoring sequence into the PLEX expression plasmid, the PLEX expression system was used to express the vaccinia viral antigen A27L on the surface of S. gordonii. Construction of LEXL 1:bh4xcrr. To facilitate the use of an additional EcoRI site on the plasmid for cloning purposes, while retaining the functionality of the rgg/gtfG sequences, plasmid pLEX1.1 :b/}4xc/τwas generated by a site-directed mutation of an EcoRI site located within the 3' region of rgg. Site-directed mutagenesis was conducted using a QuickChange XL Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA) according to the manufacturer's protocol. A silent T860C nucleotide mutation was introduced in rgg using pLEXI .0:bh4xcrr as template and primers EcoRI-F (5'-

CAAAGAACAATTTGAGCGAATCCAACTAACAGT-3'; SEQ ID NO: 11 ) and EcoRI- R (5'-TCTGCAACTACTGTTAGTTGGATTCGCTCAAAT-3'; SEQ ID NO: 12). Parental plasmid was digested using Dpn\ and products were transformed into E. coli XL-10 Gold cells (Stratagene). An erythromycin-resistant clone was screened for the presence of the mutation by confirming that EcoRI was unable to excise a band during a digestion reaction. This plasmid was subsequently sequenced to further confirm the mutation. The plasmid construct was designated as pLEX1.1 :bh4xcrr. Construction of LEXL 1:a27l(a). The gene encoding the vaccinia virus

Copenhagen A27L (p14) protein was generated by PCR using primers 5'- CGGGGTACCGACGGAACTCTTTTCCCC-3' (SEQ ID NO: 13) and 5'- CCGGAATTCCTCATATGGATCTGAAC-3' (SEQ ID NO: 14) (the underlined sequences denote, respectively, the Kpnl and EcoRI restriction sites incorporated into the primer) and vaccinia virus Copenhagen DNA as a template. The PCR product was subcloned into pCR2.1 (Invitrogen) forming plasmid pCR2.1 :A27L. Subsequent digestion of plasmid pCR2.1 :A27L with restriction enzymes EcoRI and Kpnl produced a 286 bp fragment that was ligated into EcoRI- and Kpnl -digested SPEX cloning vector pSMB104. Ligation in this manner situated the a27l structural gene, lacking a stop codon, immediately upstream and in frame with the anchoring region from the S. pyogenes M6 protein. This anchored version of the a27l gene was designated a27l(a). The DNA sequence of the A27L-coding regions of pSMB104:a27/ aJ was confirmed by sequencing. Using pSMB104:a27/(aJ as template, a PCR product was generated that possessed the a27l(a) gene flanked by Kpn\ and Nru\ restriction sites using forward primer LB37 (5'- CCGGGTACCTGGACGGAACTCTTTTC-3'; SEQ ID NO: 15), and reverse primer ML11 (5'-CGGCCGTCGCGATTAGTTTTCTTCTTTGCGTTA-3'; SEQ ID NO: 16). This product was digested with Kpn\ and Nru\, and a 1.0 kbp band was isolated. pLEXI .1 :bh4xcrr was digested with Kpn\ and Nru\ to excise the b xcr gene. A 9.8 kbp band, isolated from this reaction, was ligated to the 1.0 kbp Kpnl- and Nru\- digested PCR product, and the sequence of the inserted region was confirmed by DNA sequencing. The sequence of this plasmid, designated pLEXI .1 :a27l(a) (SEQ ID NO: 2). S. gordonii (strain GP251 ) was transformed with pLEXI Λ :a27l(a) using a synthetic competence-stimulating peptide (CSP), N-DVRSNKIRLWWENIFFNKK- COOH (SEQ ID NO: 25). Briefly, GP251 were 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 cells were frozen at -80 C in 100 μL aliquots. To transform S. gordonii, cells were quickly thawed, and 1 μg DNA and 900 μL THY were added. Cells were incubated at 37^°C for three hours and were plated out on BHI agar containing 5 μg erythromycin/mL. Following selection for erythromycin-resistant transformants, a flow cytometric analysis was used to evaluate the presence of A27L on the surface of S. gordonii. S. gordonii strains GP251 , SRL21 (which expresses A27L on its surface using the SPEX system), and a pLEX1.1 :a27l(a) transformed clone (designated SRL45) were grown to an OD₆so = 0.5. 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). Parental S. gordonii strain GP251 exhibited a low background level of fluorescence (mean fluorescence channel (MFC) = 2.3). Substantial shifts to greater fluorescence channels were observed for both SRL21 (MFC=13.3) and SRL45 (MFC=23.2). These findings suggest that the viral antigen A27L had been successfully expressed and anchored on the surface on SRL45 using the PLEX system. Discussion. Because multiple heterologous products have been successfully produced using E. coli, production of the streptococcal vaccine candidate BH4XCRR was initially attempted using this bacterium. Although BH4XCRR can be expressed using E. coli, we 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 particular products using E. coli can result in insufficient expression, expression of insoluble proteins into inclusion bodies, or an inability to express product in an intact form. Additional concerns arise from the use of E. coli as an expression vector, because of the potential for traces of lipopolysaccharide (LPS) to be present. LPS is a potent toxin and immunostimulant that can be unintentionally co-purified along with the desired product. The Gram-positive bacterium S. gordonii has been used to express numerous heterologous products, including derivatives of the S. pyogenes M protein such as BH4XCRR. Using the SPEX system, heterologous sequences are chromosomally integrated and production is driven from the native P2 promoter. Secretion is accomplished by fusion of the product with the secretion-signal sequence derived from the emmβ.1 gene from S. pyogenes. The SPEX system has been very successful for the production of particular antigens, and initial attempts at producing BH4XCRR were encouraging, particularly with regard to expression of an intact product. The yields of BH4XCRR, however, were not sufficient for the intended applications. Thus, PLEX was developed as an enhanced expression system. Instrumental to the approach was the observation that S. gordonii secretes a high-molecular weight protein under a variety of culture conditions. This protein was regularly detected as the predominant protein band in SDS-PAGE analyses of culture supematants. N-terminal sequence analysis revealed this protein as the 172 kDa glucosyltransferase, or GTF. GTF is positively regulated, at the level of transcription and also translation, by Rgg. Indeed, over-expression of Rgg, using plasmid complementation, upregulates GTF activity by approximately six-fold. The expression system described herein was designed to drive expression of BH4XCRR or other protein or polypeptide of interest from the gtfG promoter. Coding regions for the gtfG positive regulator, Rgg, were included to enhance activity at the gtfG promoter and to enhance translation. Secretion of the product was accomplished by fusion of the GTF secretional signal sequence to the bh4xcrr structural gene. BH4XCRR was selected to be expressed from a plasmid with the expectation that multiple copies of these genes would facilitate enhanced production. S. gordonii were readily transformed with plasmid p\/A838 rgg/gtfG/bh4xcrr, and expression of intact BH4XCRR by transformants were confirmed using Western blot. Following 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 hydrophobic interaction chromatography followed by gel filtration. Minimal degradation of BH4XCRR was observed following this purification procedure. EXAMPLE 4 Anchoring of multiple heterologous products on the surface of S. gordonii Surface expression of vaccinia viral antigen B5R by S. gordonii. Strain SRL39 was engineered from S. gordonii strain GP251 to express a truncated version of vaccinia viral antigen B5R using the SPEX system. Using forward primer LB7 (5'-CCGGGTACCATGACTGTACCCACTATGAATAAC. SEQ ID NO: 17, the underlined region encodes a Kpn\ restriction site) and reverse primer LB9 (5'- CCGGTCGACTGCTTCTAACGATTCTATTTC. SEQ ID NO: 18; the underlined region encodes for a Sal\ restriction site) and vaccinia virus Copenhagen DNA as template, a PCR product containing the coding sequence for B5R amino acids 22- 276 was generated. This product was subcloned into pCR2.1 to generate pCR2.1 :B5RΔ, which was digested with Kpnl and Sail. A 768 bp fragment was isolated and ligated into Kpnl- and Sa/l-digested pSMB104 to generate pSRL39. The sequence of the B5RΔ was confirmed by sequencing. S. gordonii strain GP251 was transformed with pSRL39. Transformants were screened on the basis of erythromycin resistance, and PCR was conducted to confirm the presence of the B5RΔ genetic construct. A single clone was retained and designated as SRL39. To assess surface expression of B5R, GP251 and SRL39 were grown to an OD₆₅o = 0.5. Following incubation with rabbit anti-vaccinia virus serum, adsorbed rabbit antibodies were detected using FITC-conjugated anti-rabbit IgG. The fluorescence associated with 10,000 particles was recorded and the results are presented in FIG. 8. We observed a substantial shift to higher fluorescence channels for the SRL39 sample compared to GP251. These findings were consistent with the expectations and suggest that B5R is expressed on the surface of S. gordonii strain SRL39. Construction of pLEXL3:a27l(a). In order to express multiple heterologous antigens on the surface of S. gordonii, S. gordonii strain SRL39, which expresses vaccinia viral protein B5R as an anchored product, was transformed with a pLEX plasmid bearing the gene for A27L. Because the recipient strain already possessed resistance to erythromycin, screening for pLEXI .1 :a27/ a_y)-transformed SRL39 clones would not have been feasible using erythromycin selection. Thus, pLEXI .1 :a27l(a) was modified to confer choramphenicol resistance (Cm^R). The chloramphenicol-resistance gene was PCR amplified using pAM401 as template and primers 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) and ML17 (5'-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 while the 5' sequences are homologous to the regions of pLEXI .1 a27l(a) that flank the erythromycin-resistance conferring gene. The 720 bp product from the PCR reaction was used to prime a PCR reaction using pLEXI .1 :a27/(aJ as template. Product from this reaction was digested with Dpnl to remove parental plasmid and was transformed into E. coli strain XL-10 Gold. Transformants were screened on the basis of chloramphenicol resistance and susceptibility to erythromycin. Replacement of the erythromycin-resistance gene on pLEXI .1 :a27l(a) was confirmed by DNA sequencing. This plasmid construct was designated pLEXI .3:a27/ aJ (SEQ ID NO: 3). Transformation of SRL39 with pLEXL3:a27l(a). After confirming B5R expression in S. gordonii strain SRL39, additional vaccinia viral antigen, A27L, was expressed by transforming this strain with pLEXI .Z:a27l(a). Using CSP to facilitate plasmid uptake, pLEXI .3 a27l(a) was introduced into SRL39. Transformants were screened for chloramphenicol resistance. The presence of A27L on the surface of selected clones was evaluated using flow cytometric analysis. GP251 , SRL21 , and a newly acquired clone (designated SRL46) were incubated with a mouse monoclonal antibody developed against A27L (provided by Dr. Jay Hooper,

USAMRIID, Fort Detrick, Maryland) and labeled for analysis using 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. Distributional histograms from these analyses are presented in FIG. 9. A substantial shift was observed in both SRL21 and SRL46 samples to greater fluorescence channels relative to GP251. This analysis suggests that the introduction of pLEXI .3:a27l(a) into a B5RΔ-expressing strain of S. gordonii facilitated the surface expression of an additional vaccinia viral antigen, A27L.

All cited patents and publications referred to in this application are herein incorporated by reference in their entirety for all purposes. Also incorporated herein by reference in its entirety for all purposes is priority application, U.S. Provisional Application 60/572,974 filed on May 21 , 2004.

Claims

CLAIMS We claim:

1. A nucleic acid comprising an operably linked rgg gene, a gtfG promoter, and a nucleic acid which encodes a polypeptide of interest, and wherein the polypeptide of interest is a fusion protein of a protein and a GTF signal sequence.

2. The nucleic acid of claim 1 , wherein the rgg gene, the gtfG promoter, and/or the GTF signal sequence are derived from Streptococcus.

3. The nucleic acid of claim 2, wherein the rgg gene, the gtfG promoter, and the GTF signal sequence are derived from Streptococcus.

4. The nucleic acid of claim 2, wherein the Streptococcus is S. gordonii.

5. The nucleic acid of claim 1 , wherein the polypeptide of interest comprises an antigen, an antibody or fragment thereof, an immunogenic polypeptide, a pharmaceutical agent, or an enzyme.

6. The nucleic acid of claim 1 , wherein the nucleic acid encoding the polypeptide of interest further comprises a sequence encoding an LPXTG (SEQ ID NO: 24) anchoring motif at the carboxy terminus of the polypeptide of interest, wherein X can be any amino acid.

7. The nucleic acid of claim 1 , wherein the polypeptide of interest is BH4XCRR.

8. The nucleic acid of claim 1 , wherein the nucleic acid encoding the polypeptide of interest is operably joined to a nucleic acid encoding a purification polypeptide, and wherein the purification polypeptide contains a cleavage site.

9. A vector comprising the nucleic acid of claim 1 , wherein the nucleic acid is operably inserted in said vector.

10. The vector of claim 9, wherein the vector comprises Escherichia and/or Streptococcus replicons.

11. The vector of claim 9, comprising a polylinker and/or an antibiotic selection gene.

12. A host cell transformed with the vector of claim 9.

13. The host cell of claim 12, further comprises a surface protein expression (SPEX) vector.

14. The host cell of claim 12, wherein the host cell is a Streptococcus bacterium, an Escherichia bacterium, a Lactococcus bacterium, or a Lactobacillus bacterium.

15. The host cell of claim 14, wherein the Streptococcus bacterium is S. gordonii.

16. A polypeptide of interest synthesized from the host cell of claim 12.

17. A substantially purified polypeptide of interest synthesized from host cells of claim 12.

18. A substantially purified polypeptide of interest synthesized from host cells of claim 13.

19. A substantially purified polypeptide of interest synthesized from host cells of claim 15.

20. A method of synthesizing a heterologous polypeptide of interest in a host cell comprising: (a) operably linking the nucleic acid of claim 1 to a vector; (b) inserting the vector in the host cell; (c) culturing the host cell expressing the nucleic acid under conditions wherein the polypeptide of interest is synthesized; and (d) isolating and purifying the polypeptide of interest.

21. The method of claim 20, wherein the host cell is Streptococcus or

Escherichia.

22. The method of claim 21 , wherein the host cell is S. gordonii.

23. The method of claim 20, further comprising the step of co- transforming the host cell with a surface protein expression (SPEX) vector comprising a second polypeptide of interest.