Development of PLEX, a plasmid-based expression system for production of heterologous gene products by the gram-positive bacteria Streptococcus gordonii.
While Escherichia coli expression systems have been widely utilized for the production of heterologous proteins, these systems have limitations with regard to the production of particular protein products, including poor expression, expression of insoluble proteins into inclusion bodies, and/or expression of a truncated product. Using the surface protein expression (SPEX) system, chromosomally integrated heterologous genes are expressed and secreted into media by the naturally competent gram-positive organism Streptococcus gordonii. After E. coli turned out to be an inappropriate expression system to produce sufficient quantities of intact product, we successfully utilized SPEX to produce the heterologous antigen BH4XCRR that is designed from sequences homologous to the S. pyogenes M-protein C-repeat region. To further enhance production of this product by S. gordonii, we sought to develop a novel system for the production and secretion of heterologous proteins. We observed that under various growth conditions, S. gordonii secreted high levels of a 172 kDa protein, which was identified by N-terminal sequence analysis as the glucosyltransferase GTF. Here we report on the development of a plasmid-based expression system, designated as PLEX, which we used to enhance production of BH4XCRR by S. gordonii. A region from the S. gordonii chromosome that contains the positive regulatory gene rgg, putative gtfG promoter, and gtfG secretion-signal sequence was cloned into the E. coli/Streptococcus shuttle plasmid pVA838. Additionally, the bh4xcrr structural gene was cloned into the same plasmid downstream and in-frame with rgg and gtfG. This plasmid construct was transformed into S. gordonii and BH4XCRR was detected in culture supernatants from transformants at greater concentrations than in supernatants from a SPEX strain expressing the same product. BH4XCRR was easily purified from culture supernatant using a scalable two-step purification process involving hydrophobic-interaction and gel-filtration chromatography.
Warren
Travis K
TK
Siga Technologies, Inc., 4575 SW Research Way, Suite 230 Corvallis, OR 97333, USA.
Lund
S Amanda
SA
Jones
Kevin F
KF
Hruby
Dennis E
DE
D000942:Antigens, Bacterial / Q000235:genetics* / Q000302:isolation & purification; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D010957:Plasmids / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D013291:Streptococcus / Q000737:chemistry*
D000942:Antigens, Bacterial