Molecular cloning and expression of a Streptococcus mutans major surface protein antigen, P1 (I/II) , in Escherichia coli

Lee, S.F.; Progulske-Fox, A.; Bleiweis, A.S.

Infection and Immunity 56(8): 2114-2119


ISSN/ISBN: 0019-9567
PMID: 3135272
Accession: 040723235

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Antigen P1, also called I/II, is one of the most abundant cell wall proteins of the mutans streptococci. It has been suggested that P1 may be involved in cell adherence to tooth surfaces and in sucrose-induced cell aggregation. As a first step toward fully understanding its biological functions, the P1 gene, which has been designated spaP1, from Streptococcus mutans NG5 (serotype c) has been cloned into Escherichia coli JM109 by a shotgun procedure with pUC18 as the vector. The recombinant strain expressing P1 carries a 5.2-kilobase DNA insert whose restriction map has been determined. This map is completely different from that of spaA of Streptococcus sobrinus (serotype g), even though P1 and SpaA are antigenically related. Southern hybridization revealed that DNA sequences closely homologous to spaP1 were present in serotypes c, e, and f, and similar sequences also existed in strains of serotypes a and d. The expression of the cloned spaP1 was found to be independent of the lac inducer and the orientation of the DNA insert, suggesting that it carries its own promoter. Western blotting (immunoblotting) revealed at least 20 bands reacting with a mixture of three anti-P1 monoclonal antibodies. The highest-molecular-weight reactive band was comparable in size to the parent P1 (185 kilodaltons [kDa]); however, the major reactive bands were smaller (approximately 160 kDa). Expression of cloned P1 in E. coli LC137 (htpR lonR9) resulted in the increased prominence of the 185-kDa protein reactive band. Ouchterlony immunodiffusion showed partial identity between the parent and cloned P1. In E. coli, P1 was detected primarily in the periplasm and extracellular fluid.