Chromatographic Study of Spirosin by Use of Monoclonal Antibodies to Spirosin of Yersinia enterocolitica

Abstract

Two monoclonal antibodies (MAbs) reacting with spirosins from Enterobacteriaceae were obtained in a course of screening MAbs to spirosin from Yersinia enterocolitica . The antibodies were designated MAbs-S44 and S50. They were IgG2b and IgG2a,respectively, both with lc light chains. On Western blotting after limited proteolysis of YE72 spirosin with Staphylococcus aureus V8 protease, they reacted markedly with peptide fragments of 27 and 35 kDa, suggesting the presence of an antigenic determinant on the fragments. When supernatant cell lysate from Escherichia coli K12 was chromatographed on DEAE-cellulose and Sepharose CL-6B columns successively, a 96-kDa protein with alcohol dehydrogenase (ADH) activity was always associated with reactivity to MAb-S50. These findings combined with N-terminal amino acid sequences clearly indicate the identity of spirosin to ADH in E. coli.

Purification of spirosin from Lactobacillus reuteri DSM 20016 and partial characterization based on its amino acid sequences.

“…Recently, the spirosome of Escherichia coli has turned out to be an anaerobically-induced multifunctional alcohol dehydrogenase (ADH) (Imamura et al, 1992;Kessler et al, 1991;Yamato et al, 1995). However, the function of spirosome, especially in Lactobacillus, has not been clarified yet probably because Lactobacillus spirosin has never been isolated in a pure form appropriate for biochemical study.…”

“…The spirosome from Lactobacillus and Enterobacteriaceae is composed of 95 kDa protein subunits called spirosin (Ueki et al, 1982;Yamato et al, 1995). There have been many reports on morphological, biochemical and immunochemical studies of spirosome as quoted in our previous papers (Yamato et al, 1994(Yamato et al, , 1995.…”

“…Likewise, the sequence of the 35 kDa fragment by LEP has 46% identity and 86% similarity to that of the 224 to 238 residues of the same protein. It is reasonable that the two peptide fragments of spirosin have considerable sequence homology to E. coli ADH (E. coli spirosin as mentioned above), because spirosins from L. reuteri and L. acidophilus cross-reacted with those from Enterobacteriaceae with regard to MAb-S44 which was raised against the spirosin of Yersinia enterocolitica (Yamato et al, 1995). On the other hand, the sequence of the 25 kDa fragment by V8P has 53% identity and 61% similarity to O171, the sequence in an open reading frame of E. coli corresponding to 171 amino acids, which is 73% identical to 161 residues of the outer membrane protein C precursor (porin) from Salmonella typhi.…”

“…The cell lysate was applied to a column of DEAE-cellulose (DE52; Whatman BioSystems Ltd., Maidstone, Kent, UK), equilibrated with 25 mM Tris-HCl, pH 8.3, and eluted with 0 to 0.5 M KCl in the same buffer. Each column fraction was assayed both for ADH activity and reactivity with monoclonal antibody (MAb)-S44 by EIA as described previously (Yamato et al, 1995). As shown in Fig.…”