Surface Expression of a Protective Recombinant Pertussis Toxin S1 Subunit Fragment in Streptococcus gordonii

Schematic diagram showing the construction of thespaP-s1 fusion gene. The S1 gene (coding for amino acid residues 2 to 233 of the mature S1) was amplified by PCR from the PT operon carried on pPTX42 (E. coli ATCC 67046 [14]) by standard methods (19) using the primers 5′-GATCCTCCCGCCACCGT-3′ and 5′-GGATCGATAACGAATACGCGATGCT-3′. (The underlined bases are added sequence for a ClaI site.) The amplicon was treated with Klenow fragment, restricted with ClaI, purified from agarose gels with a Gene-Clean kit (Bio 101, La Jolla, Calif.), and ligated into the EcoRV-ClaI sites of pN1C4, a pUC18 derivative carrying the 3′ DNA of spaP from S. mutans, coding for the C-terminal 144 amino acids containing the surface protein anchoring domain of SpaP (8). The ligated DNA was transformed into competent E. coli HB101, and the resulting plasmid was designated pPTS1. To provide the fusion gene with the spaP promoter, the 1.5-kbSmaI-XbaI fragment from pPTS1 was cloned into theEcoRV-XbaI sites of pSMI/II, a pUC18 derivative carrying the complete spaP gene (10). The resulting plasmid isolated from one of the E. coli HB101 transformants was named pRJMI. The 5.6-kbKpnI-ScaI fragment from pRJMI was further cloned into pDL276, creating pRJMII. pRJMIII was further constructed by ligating the 10.0-kb NruI-KpnI fragment from pRJMII to the 4.5-kb EcoRV-KpnI fragment from pSMI/II.