Over-expression and Purification of Formate Dehydrogenase form Pichia pastoris in Escherichia coli
- VernacularTitle:毕赤酵母甲酸脱氢酶在大肠杆菌中的高表达及纯化
- Author:
Shao-Xin CHEN
;
Bing-Zhao SHI
;
- Publication Type:Journal Article
- Keywords:
Pichia pastoris;
Formate dehydrogenase;
Gene expression;
Purification
- From:
Microbiology
1992;0(01):-
- CountryChina
- Language:Chinese
-
Abstract:
Formate dehydrogenase(FDH)coding gene was amplified from genomic DNA of Pichia pastoris by polymerase chain reaction, and the codon TAG(bases 649-651)was mutated to GAG using site-directed mutagenesis.The recombinant plasmid pET-FDH was con- structed by inserting the mutated DNA fragment into expression vector pET-22b(+),and transformed into E.coli BL21(DE3).FDH was expressed as a form of soluble prutein fused with 6?His tag at high level through IPTG induction.The amount of FDH was up to about 30% of the total cell protein.The cells-free crude extract was purified by one affinity chromatographic step,and resulting enzyme preparation revealed a specific activity of 6.45 U/mg.
