利用S-层蛋白在细胞表面展示α-淀粉酶和金属硫蛋白

Cell Surface Display of α-amylase and Metallothionein by Using S-layer Protein of Bacillus thuringiensis Strain CTC

选用苏云金芽胞杆菌CTC菌株细胞表面S 层蛋白作为表面展示载体 ,利用其N 端信号肽以及锚定序列分别将不同生物特性的外源蛋白带到胞外并锚定在细胞的表面。为研究外源蛋白的分子量和生化特性对表面展示以及对其在细胞表面生物活性的影响 ,选用分子量较大的分泌性蛋白α 淀粉酶和富含半胱氨酸的非分泌性蛋白金属硫蛋白作为目标蛋白。将α 淀粉酶的结构基因 (amy)和金属硫蛋白的结构基因 (smtA)与S 层蛋白的锚定区slh结合 ,构建融合蛋白基因slh amy及slh smtA ,克隆至穿梭载体pHT30 4上 ,得到重组质粒pBMBSA 30 4和pBMBSM 30 4 ;转入带有帮助质粒的重组菌株BMB171 pBMB CSA中 ,得到新的重组菌株BMBSAC和BMBSMC。SDS PAGE显示融合蛋白SLH AMY和SLH SMTA在重组菌的表面得到了表达。利用锥虫蓝染色 打孔加膜法表明重组菌SAC的确具有表面酶活性 ,α 淀粉酶被固定在细胞表面。碘液法测定的数据表明重组菌SAC的菌悬液样品较之受体菌BMB171菌悬液样品酶活提高了 4 2 4 %。Ni NTA 琼脂糖微球吸附试验显示重组菌SMC能够被微球所吸附 ,证实SMTA在重组菌表面具有活性。对游离镉离子的吸附试验显示重组菌对镉离子的吸附能力是对照受体菌的 4倍。

A bacterial cell surface display system was developed by employing S-layer protein of strain CTC as a display carrier to diaplay interesting proteins with different characteristics. α-amylase and Metallothionein were selected and fused with S-layer.The two proteins were translocated on the surface of host strain by means of the signal peptide and anchoring sequences (slh) which were essential to anchor interesting proteins on the cell surface, respectively. Recombinant plasmids pBMBSA-304 and pBMBSM-304, one containing fusion gene slh/amy, and the other slh/smtA, were constructed and introduced into recombinant strain BMB171/pBMB-CSA , resulting in recombinant strain BMBSAC and BMBSMC. SDS-PAGE profiles demonstrated that the fusion protein SLH/AMY and SLH/SMTA were expressed on the surface of recombinant strain. The result of trypan blue drying indicated that α-amylase was anchored on the surface of cell and there was enzyme activity on the surface of recombinant strain. The detectable result of iodine method demonstrated that enzyme activity was increased 42.4% by comparing enzyme activity of intact cells of recombinant strain BMBSAC with that of strain BMB171. Moreover, Ni-NTA-agarose beads adhesion test showed that only recombinant strain BMBSMC could be attached to agarose beads. The result of the dissociate Cd^(2+) absorption demonstrated that absorption activity of recombinant strain was as four times as the control.