腾冲嗜酸热两面菌S5分子伴侣β亚基的表达、纯化和活性的初步分析

The Expression,Purification of Chaperonin β Subunit from the Thermoacidophilic Archaeon,Acidianus tengchongensis and its Activity Analysis

摘要:

用NdeI和BamHI酶切回收腾冲嗜酸热两面菌S5的分子伴侣β亚基基因片段插入pET-23b的相应位置,并分别在BL21(DE3)和Rosetta-gami^TMB(DE3)pLysS中表达。表达的β亚基以可溶的形式存在。β亚基在Rosetta-gami^TMB(DE3)pLysS中表达较高,其占菌体总蛋白的16．2%,且以单体和聚体形式同时存在。表达的菌体经超声破碎、70℃热处理后,上清中β亚基蛋白含量达到30%,再经(NH₄)₂SO₄沉淀、Bio-Gel A-1．5m和DEAE-Sepharose CL-6B柱层析,得到在SDS-PAGE呈电泳均一的β亚基,Native-PAGE表明其为聚体,有弱的ATPase活性。

Abstract:

The fragment of pGEM-T easyβ,containing the gene of chaperonin subunitβof Acidianus tengchongensis strain S5,was inserted into the plasmid pET23b with Ndel and BamHI,designated as pET236βand transformed into BL21(DE3)or Rosetta-gami^TMB(DE3) pLysS.The expressed protein was soluble.Expression of the subunitβwas 16.2% of total cell proteins in Rosetta-gami^TMB(DE3)pLysS and displayed both monomer and oligomer.The recombinant subunitβwas purified by means of sonication,heating at 70℃for 10 min,am- monium sulfate precipitation,chromatography on Bio-Gel A-1.5m and DEAE-Sepharose CL-6B. The purified recombinant of subunit β dis-played oligomer on native-PAGE and exhibited weak ATPase activity.