Two-step purification of Bacillus circulans chitinase A1 expressed in Escherichia coli periplasm |
| |
Authors: | Chen Chun-Ti Huang Chien-Jui Wang Yi-Huei Chen Chao-Ying |
| |
Affiliation: | Department of Plant Pathology and Microbiology, National Taiwan University, Taipei 106, Taiwan, ROC. |
| |
Abstract: | 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 microg/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. |
| |
Keywords: | |
本文献已被 PubMed 等数据库收录! |
|