Expression,purification and identification of I278T-mutant human cystathionine βsynthase
10.3969/j.issn.1673-4130.2014.09.001
- VernacularTitle:人胱硫醚β合酶I278T突变体的表达、纯化及鉴定
- Author:
Weining NIU
;
Le XU
;
Menglin YANG
;
Shanshan CAO
- Publication Type:Journal Article
- Keywords:
cystathionine beta-synthase;
mutation;
particle size;
Zeta potential
- From:
International Journal of Laboratory Medicine
2014;(9):1089-1091
- CountryChina
- Language:Chinese
-
Abstract:
Objective To investigate the expression and purification I278T-mutant human cystathionineβsynthase(CBS) in E . coli .Methods Site-directed mutagenesis by overlap extension using the polymerase chain reaction (PCR) was employed to construct mutant plasmids pGEX4T-1-CBS(I278T) ,which was induced and expressed in a medium containing 3% ethanol ,purified by affinity chromatography to obtain mutated CBS (I278T) protein .The activity ,UV-visible absorption spectroscopy ,protein particle size and Zeta potential of the purified protein were measured .Results Plasmid pGEX4T-1-CBS(I278T) was successfully constructed .The yield ,the specific activity and activity recovery of purified mutant CBS (I278T ) protein were 2 .3 mg/L ,21 .4 U/mg and 22 .6% .S-adenosylmethionine(AdoMet) with final concentration of 1 mmol/L showed no activation toward mutant CBS (I278T) protein .Ac-cording to UV-visible absorption spectroscopy analysis ,purified mutant CBS(I278T) had characteristic absorption peaks at 429 nm and 550 nm for heme-binding proteins .Protein average particle size was 7 .5 -10 .1 nm ,mainly in the form of tetramers ,and Zeta potential was - 16 .3 mV .Conclusion The methods of expression ,purification and identification of I278T-mutant human cystathionineβsynthase in E .coli were successfully established .
