Molecular Cloning of S-Layer Protein Gene from Bacillus Thuringiensis.

Extraction of S-layer protein by treatment with a 6M urea indicated that there appeared to be extra-high molecular weight protein in the extracts obtained from Bacillus thuringiensis subsp. israelensis (B.t.i.) strain 4Q2. The antibody toward this large molecular weight protein was prepared and used for locating of S-layer protein on B.t.i. by using indirect immunofluorescent technique. Immunodiffusion reaction and Western blot analysis confirmed the specificity of the anti-S-layer protein. It was found that the antibody against S-layer protein inhibited the plasmid transfer via the conjugation-like process. The frequency of transfer of plasmid pBC16 was found to be 9.7×10-6 and less than 10-8 in the presence and absence of anti-S-layer antibody, respectively. Using antibody detection technique, S-layer protein gene from B.t.i. strain 4Q2 was cloned in phagemid pKSII and plasmid pUC12 of E.coli DH5α. Three positive clones containing the gene encoding for the S-layer protein were obtained. Two clones had the insert of 5.2 kb, and the other one had an insert of 6.6 kb. The expression of the S-layer protein gene was detected by using Western blot analysis. [ABSTRACT FROM AUTHOR]