Immunochemical studies on expression of quinoproteins in Escherichia coli

Abstract

An immunochemical method has been developed as the most sensitive tool for studying the expression of quinoproteins containing pyrroloquinoline quinone (PQQ) in E. coli. The PQQ was conjugated to bovine serum albumin (BSA), and the conjugant was purified by using a KwikSep(TM) dextran desalting column chromatography. The PQQ-BSA conjugant was immunized to rabbits, and the IgG fractions of the antisera were purified. The most sensitive antibody againt PQQ-BSA conjugant recognized some nanogram quantity of the antigen on the blot, but had little cross reactivity with BSA. Using this batch of the antibody, all the immunochemical assays of quinoproteins in E. coli were performed, Some six different PQQ-specific spots were detected by Western blot analysis of the soluble proteins in E. coli after two-dimensional gel electrophoresis. Their molecular weights on the blot were estimated to be about 100-, 90-, 72-, 58-, 52-, and 50-kDa. Their pI values fell in the range from 4.8 to 5.5. These results strongly suggest that quinoproteins are present in E. coli, and that the protein moieties were covalently bound to PQQ.