摘要
PLZF(promyelocytic leukaemia zinc finger protein)是一种重要的转录抑制因子,它由位于N端的BTB结构域和C端的锌指结构域构成。鉴于目前对于锌指结构域的立体结构还不是十分清楚,对其进行了高效表达和提纯。为了表达PLZF蛋白的锌指结构域,在其编码序列的5'端加上起始密码ATG后插入到表达载体PET-11a的多克隆位点。构建好的表达质粒转化到BL21(DE3)大肠杆菌内并用IPTG诱导表达,发现重组蛋白主要以不溶性的包涵体形式在胞内表达。用含有SDS变性剂的缓冲液溶解包涵体后,采用凝胶过滤方法将重组蛋白纯化到纯度达96%以上。对纯化后的蛋白质用反透析的方法进行复性,然后用DNA结合实验进行活性分析,发现复性后的蛋白质具有特异的DNA结合活性,这为进一步研究PLZF蛋白锌指结构域的立体结构打下了重要基础。
PLZF is an important transcription repressor.It consists of an N-terminal BTB domain and a C-terminal zinc finger domain.To date,the three dimensional structure of the zinc finger domain is still not clear.Hence,expression and purification studies were performed.To produce the zinc finger domain of PLZF protein,the sequence encoding zinc finger domain fragment with a start codon added to the 5′ was inserted into PET-11a expression vector.The expression plasmid was transformed into E.coli BL21（DE3） strain and protein expression was induced by IPTG.The recombinant protein was found to be expressed as inclusion bodies.The inclusion body was solubilized using buffers containing SDS detergent and proteins were purified to more than 96 % homogeneity through gel filtration.The purified proteins were denatured and then refolded by reverse dialysis.The refolded proteins were characterized by DNA electrophoretic mobility shift assay（EMSA） and found to be active.An important foundation has been laid for further three-dimensional structural study on the zinc finger domain of PLZF.
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
2011年第11期1-5,共5页
China Biotechnology
基金
上海市自然科学基金资助项目(11ZR1416800)
