[Cloning and expression of an Aspergillus fumigatus chitosanase gene].
According to published DNA sequence of Aspergillus fumigatus chitosanase(Csn) gene, 8 long single DNA strands each about 100bp and 4 DNA primers were designed and synthesised. By PCR, 8 DNA strands were connected into a complete chitosanase gene of 624bp. This chitosanase gene was not identical with its wild type, some point mutations were introduced into its DNA sequence by special design of those 8 DNA strands. These mutaions did not change amino acid composition of the chitosanase, however, the codens were changed into E. coli favorites. The Csn gene was cloned into plasmid pGEM-Teasy and verified by DNA sequence analysis. Thereafter, Csn gene was subcloned into a fusion-protein expressing vector pGEX-3X. Recombinant plasmid pGEX-Csn was transformed into E. coli DH5alpha and the transformant was induced expressing with 0.5 mmol/L IPTG. Expressing product was analyzed by SDS-PAGE, fusion protein GST-Csn was purified by affinity chromatography. By factor X a digestion GST-Csn was cleaved and GST was taken out by another chromatography. The biological activity of recombinant chitosanase(rCsn) was also detected, as a result the recombinant Csn had a strong ability of degrading chitosan, which was much higher than lysozyme. Its chitosan-degradation activity could be influenced by pH and temperature.
Liang
Dong-Chun
DC
Institute of Endocrinology of Tianjin, Tianjin 300070, China. liangdongchun1225@263.net
Zuo
Ai-Jun
AJ
Guo
Gang
G
Zhang
Jing-Yu
JY
D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification
D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C019868:chitosanase