摘要
以NCBI报道的Comamonas testosteroni ATCC11996中3α-羟类固醇脱氢酶基因(3α-hydroxysteroid dehydrogenase gene,3α-hsd,AF092031.2)为模板,通过改变碱基序列但不影响酶的氨基酸序列,将该基因相对于大肠杆菌的密码子适用指数由0.78提高到0.87,GC含量由原来的63.17降低到56.46,大幅度减少了GC簇及寡聚A和T局域的存在,提高3α—hsd的可表达性。全合成基因定向克隆到pET28a载体中,并转化至大肠杆菌B121(DE3)中表达。采用乳糖诱导后,SDS—PAGE检测在约27 kD处有一高效表达的蛋白条带。采用Ni螯合柱分离纯化3α-HSD,获得了较高纯度及较高的产率。酶学性质初步分析表明,全基因合成表达的3α-HSD的性质与原始的3α-HSD基本相同。
In this research, new 3α-hsd was designed and synthesized on the basis of 3ot-hsd sequence （AF092031.2）. The contents of GC fell from 63.17 to 56.46 and the codon applicable index increased from 0.78 to 0.87. Besides, the existence of continuous GC, continuous A and T were reduced. The synthesized 3a-hsd was subcloned into expression vector pET28a（ ＋ ） to construct the recombinant expression vector pET-28a-HSD. After lactose induction, soluble 3α-HSD was over-produced by E. coli BL21 （DE3） harboring the expression constructs. Recombinant 3α-HSD purified by Ni- NTA showed a single band about 27 kD on SDS-PAGE gel. The specified activity was about 5 U/mL and the activity recovery was 64.1%.
出处
《工业微生物》
CAS
CSCD
2014年第1期34-39,共6页
Industrial Microbiology
基金
江苏省科技计划项目(SBE201170578)(No.SBE201170578)
江苏省科技计划项目(SBE201077545)(No.SBE201077545)
江苏省自然科学基金(SBK201240329(No.SBK201240329)
国家高技术研究发展计划(863计划)(2012AA021201)
