A Simple Purification Method of Vibrio cholerae Non‐O1 Hemagglutinin/Protease by Immunoaffinity Column Chromatography Using a Monoclonal Antibody

Abstract

A new simple purification method (I) for Vibrio cholerae non-O1 hemagglutinin/protease (NAG-HA/P) was developed. The method (I) requires only an immunoaffinity column chromatography using a monoclonal antibody against NAG-HA/P. The method (I) is much simpler than previously reported purification method (II) (Honda, T. et al, Infection and Immunity 57: 2799-2803) which required four or more complicated chromatographic procedures. Method (I) also gave an improved recovery rate (about 27%) compared with (II). The molecular weight of NAG-HA/P purified by method (I) was mainly 34 kilodaltons (kDa) with a little of 32 kDa, whereas that of NAG-HA/P purified by (II) was usually 32 kDa. Immunological analysis by the Olichterlony double gel diffusion test and Western blotting test using polyclonal antibody against 32 kDa protein revealed that the 34 and 32 kDa proteins are immunologically indistinguishable and thus it is supposed that 34 K protein is an isoform or a preform of the 32 K protein.Vibrio cholerae non-O1 (NAG vibrio), which is similar to V. cholerae O1, but is not agglutinated by V. cholerae O1 antiserum, is an important cause of human diarrheal diseases. Gastroenteritis associated with V. cholerae non-O1 shows various clinical symptom (2, 12), such as abdominal pain, mucous and bloody diarrhea as well as watery diarrhea similar to cholera due to V. cholerae O1. These various symptoms may be explained by the production of a variety of toxins by V.

Vibrio cholerae O139 produces a protease which is indistinguishable from the haemagglutinin/protease of Vibrio cholerae O1 and non-O1

“…The crude 0139-P was applied to a column (1 X 7 cm) packed with mAb 2-4-2-coupled Sepharose 4B [15] and the column was washed with 0.01 M phosphate buffer (pH 7.0) until the absorbance of the eluate at 280 nm reached the baseline to remove non-specific materials. Specifically bound material (0139-P) was eluted with 0.2 M glycine-HCl buffer (pH 2.7) containing 0.5% (w/v) NaCl and was immediately neutralized with 2.0 M Tris.…”

“…The culture supemates of B1854 and TH81 were fractionated with 40-55% (w/v> ammonium sulfate and extensively dialyzed against 0.01 M phosphate buffer (pH 7.0) at 4"C, and used as crude samples of 0139-P and NAG-HA/P. The crude samples were further purified by im-munoaffinity chromatography using a monoclonal antibody against NAG-HA/P as described previously [15].…”

In vitro proteolytic processing and activation of the recombinant precursor of El Tor cytolysin/hemolysin (pro-HlyA) of Vibrio cholerae by soluble hemagglutinin/protease of V. cholerae, trypsin, and other proteases

“…Purification of soluble HA/protease. Soluble HA/protease was purified from V. cholerae non-O1 TH81 by ammonium sulfate precipitation and immunoaffinity column chromatography as previously described (15).…”