化学渗透法破碎基因重组E.coli提取重组人SOD包含体和复性

Study on Chemical Permeabilization Agents Disrupting the Recombinant E.coli Cell to Release rhSOD Inclusion Body and Renaturation

以化学渗透法破碎重组大肠杆菌细胞释放重组人超氧化物歧化酶(rhSOD)包含体并对其复性。选择四种化学试剂(Triton X-100、EDTA、NaOH和SDS)对rhSOD重组大肠杆菌细胞进行破碎实验,以SDS-PAGE检测破菌效果,结果0.5%Triton X-100和10 mmol·L-1 EDTA可以较好地破碎重组大肠杆菌细胞,所提取的包含体溶解液纯度与超声法相近,可达65%左右。采用透析法复性包含体,尿素终浓度0.1 mol·L-1时rhSOD包含体复性效果优于1.0 mol·L-1,比活分别为:Triton法2800 U·mg-1,EDTA法3200 U·mg-1,超声法2800 U·mg-1,复性rhSOD纯度达85%左右。本研究建立的用于破碎重组大肠杆菌释放rhSOD包含体的Triton法和EDTA法,包含体复性后比活等于或高于超声法,与机械破菌法相比,简便有效,成本低廉,无需特殊破菌设备,为放大规模破菌提取重组大肠杆菌包含体和复性奠定了实验基础。

The recombinant E.coli cell was disrupted with chemical permeabilization agents,such as Triton X-100,EDTA,NaOH and SDS,to release and renature rhSOD inclusion bodies.The disrupting effects were analyzed by SDS-PAGE and the inclusion bodies were renatured by dialysis.The results show that 0.5% Triton X-100 and 10 mmol·L-1 EDTA can effectively disrupt recombinant E.coli cell.The purity of the inclusion bodies treated with 0.5% Triton X-100 or 10 mmol·L-1 EDTA is about 65%,which is similar to that prepared by ultrasonic.The renaturation efficiency of dialysis with final urea concentration of 0.1 mol·L-1 is better than that of dialysis with final urea concentration of 1 mol·L-1.The renatured rhSOD purity is about 85% and the enzyme activity is 2800 U·mg-1（by TritonX-100）,3200 U·mg-1（by EDTA） and 2800 U·mg-1（by ultrasonic）,respectively.Comparing with the mechanical disrupted method,the way of using chemical permeabilization agents is simple and efficient.It gives an experimental foundation for disrupting recombinant E.coli cell and preparing inclusion body on a large scale.