生物技术通报2016，Vol.32Issue(9)：246-252,7.DOI:10.13560/j.cnki.biotech.bull.1985.2016.09.033
DSPAα1缺失突变体在毕赤酵母中的表达及活性研究
Expression and Activity Analysis of DSPAα1 Deletion Mutants in Pichia pastoris
陈韵 1牛纯青 1宋小双 1苏畅 1华子春 2刘堰1
作者信息
- 1. 重庆西南大学生命科学学院，重庆 400715
- 2. 南京大学医药生物技术国家重点实验室，南京 210093
- 折叠
摘要
Abstract
There are 4 types of desmodus salivary plasminogen activators(DSPAs),i.e.,DSPAα1,DSPAα2,DSPAβ and DSPAγ. DSPAα1 and DSPAα2 contain a finger domain(F),an epidermal growth factor domain(E),a kringle domain(K)and a serine proteinase domain(P);DSPAβ contains E,K and P domains ;and DSPAγ contains K and P. The effect of DSPAα1’s structure on fibrinolytic activity was also investigated. A deletion mutant of DSPAα1(mDSPAα1)lacking the E domain was synthesized by splicing overlap extension PCR(SOE-PCR)method,and the recombinant plasmids of mDSPAα1/pPIC9K,DSPAβ/pPIC9K and DSPAγ/pPIC9K were constructed and transformed into Pichia pastoris GS115. Fibrinolytic activity was determined by fibrin plate assay. Results showed that the expression of DSPAγ was not detected,the activities of DSPAα1,DSPAβ,and mDSPAα1 were 2.64 × 105 U/mg,1.32 × 104 U/mg and 151.52 U/mg respectively. The total activity hardly changed when using DSPAα1,mDSPAα1 and DSPAβ together,but there led a 2-3 folds of reduction by using either two of them. The mDSPAα1 exhibited almost no fibrinolytic activity,whilst DSPAβ retained comparable catalytic activity to the wild-type DSPAα1. Deletion mutant studies illustrated that N-terminal region of DSPAα1 greatly affected the PA activity,and it without E domain nearly lost its activity,indicating that the E domain of DSPAα1 plays a key role during fibrinolytic process.关键词
DSPAα1/SOE-PCR/缺失突变体/毕赤酵母/纤维平板法Key words
DSPAα1/SOE-PCR/deletion mutants/Pichia pastoris/fibrin plate assay引用本文复制引用
陈韵,牛纯青,宋小双,苏畅,华子春,刘堰..DSPAα1缺失突变体在毕赤酵母中的表达及活性研究[J].生物技术通报,2016,32(9):246-252,7.
