title: SPEX, a system for the expression of recombinant proteins from gram-positive bacterial vectors.
abstract: Using a conserved pathway for surface protein extrusion, a system has been developed for the expression and secretion of proteins from gram-positive bacteria. As proof-of-concept, the Streptococcus gordonii Challis strain has been engineered to express a series of recombinant proteins fused to the conserved region of the M6 protein of Streptococcus pyogenes. In the prototype surface protein expression system, the recombinant M6 protein is anchored to the surface of S. gordonii cells expressing it. In order to overexpress the protein and easily purify it away from the bacteria, the protein was modified to enable it to be secreted into the medium. To accomplish this, a stop codon was introduced into the gene just prior to the anchor region using site-directed mutagenesis. Using enzyme-linked immunosorbent assays, it was possible to quantitate the amount of protein expressed using this system. With little or no optimization, 3 mg of protein per liter of culture was expressed and secreted into the medium of a bacterial culture grown to an OD600 equal to 1.0. This system should be broadly applicable for the expression and secretion of a variety of proteins (antigens, hormones, and enzymes) directly into the medium.
authors.affiliation: Department of Microbiology, Oregon State University, Corvallis, Oregon, 97331-3804, USA.
authors.forename: D M
authors.initials: DM
authors.lastname: Myscofski
authors.forename: D E
authors.initials: DE
authors.lastname: Hruby
mesh_terms: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics*; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016622:Silver Staining; D013291:Streptococcus / Q000378:metabolism*; D013728:Terminator Regions, Genetic; D014169:Transformation, Bacterial
chemical_list: D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D003062:Codon; D011993:Recombinant Fusion Proteins; C014689:streptococcal M protein