Two-step purification of Bacillus circulans chitinase A1 expressed in Escherichia coli periplasm

A protein purification procedure was developed to efficiently and effectively purify the target enzyme, chitinase A1 of Bacillus circulans WL-12, from Escherichia coli DH5alpha carrying the chiA gene with its natural promoter in the plasmid pNTU110. Chitinase A1 was purified to apparent homogeneity from E coli periplasm with a final recovery of 90.6%. Two main steps were included in this protein purification procedure, ammonium sulfate precipitation (40% saturation) and anion-exchange chromatography at pH 6.0 using Q Ceramic HyperD column. The yield of chitinase A1 was estimated at 95 mug/L. A polyclonal antibody against chitinase A1 was raised by immunizing BALB/c mice with chitinase A1 purified from E coli DH5alpha(pNTU110). As indicated by Western blot analysis, a 3000-fold diluted antibody detected purified chitinase A1 from E coli DH5alpha(pNTU110) in an amount of at least 1 ng and specifically detected chitinase A1 produced by B. circulans WL-12. (C) 2004 Elsevier Inc. All rights reserved.