Hydrophobicity engineering to facilitate surface display of heterologous gene products on Staphylococcus xylosus

CD4+T-Cell-Mediated Antiviral Protection of the Upper Respiratory Tract in BALB/c Mice following Parenteral Immunization with a Recombinant Respiratory Syncytial Virus G Protein Fragment

“…Gene assembly, vector constructions, expression, and first-step protein purification of BBG2Na and BBG2⌬Ca (BBGnat and BBGcys, respectively, in reference 37) were undertaken as previously described (37). Gsera was derived from G2Na by alternative PCR site-directed mutagenesis (30), such that the conserved Cys residues at positions 173, 176, 182, and 186 were each mutated to Ser. Six deletion mutants were generated by PCR from a G2Na template using a single 5Ј oligonucleotide (5Ј-CGA GAA TTC CAT GCA GAC CCA GCC GAG-3Ј), incorporating a unique EcoRI site (underlined), and a series of nested 3Ј oligonucleotides (5Ј-ATCAAGCTTATTTGTTCGGGATAC-3Ј, 5Ј-ATCAAGCTTACGGTTTTTT GTTCGGGATACG-3Ј, 5Ј-ATCAAGCTTATTTGCCCGGTTTTTTGTTC-3Ј, 5Ј-ATCAAGCTTAGGTTTTTTGCCCGG-3Ј, 5Ј-ATCAAGCTTAGGTCGTG GTTTTTTGCCCG-3Ј, and 5Ј-ATCAAGCTTACGGGATGGTTTTGC-3Ј), each incorporating a unique HindIII site (underlined).…”

Surface display of a functional single-chain Fv antibody on staphylococci

“…DNA sequencer system (Pharmacia Biotech, Uppsala, Sweden). A verified clone was SalI-HindIII subcloned to the expression vector pSEmp18ABPXM (29), used in S. xylosus, or pSPPmABPXM (38), used in S. carnosus. The vectors, obtained, designated pSE␣IgEABPXM and pSPP␣Ig EABPXM, respectively, expressing the recombinant receptors were used to transform staphylococcal protoplasts.…”

“…The gene fragment encoding the scFv was inserted into the two general expression vectors pSEmp18ABPXM (29) and pSPPmABPXM (38), designed for surface display on S. xylosus and S. carnosus, respectively. The two resulting expression vectors were designated pSE␣IgEABPXM (Fig.…”

“…Lipoprotein-assisted surface display has also been accomplished in Mycobacterium bovis BCG (46). Various gram-positive bacteria, such as staphylococci (16,29,30,38), streptococci (15,34,35,37), and enterococci (15), have recently started being investigated for surface display purposes. For gram-positive bacteria, cell surface anchoring has been achieved by using C-terminal regions of different gram-positive receptors, including Staphylococcus aureus protein A (16,29,30,38), the Streptococcus pyogenes M6 protein (34,35), and the S. pyogenes fibronectin-binding protein (15).…”

“…For gram-positive bacteria, cell surface anchoring has been achieved by using C-terminal regions of different gram-positive receptors, including Staphylococcus aureus protein A (16,29,30,38), the Streptococcus pyogenes M6 protein (34,35), and the S. pyogenes fibronectin-binding protein (15). The most common application for bacterial surface display is the development of live bacterial vaccine delivery systems, since the cell surface display of heterologous antigenic determinants has been considered advantageous in order to induce antigen-specific antibody responses when live recombinant cells are used for immunization (9,29,44,46).…”