亲和标记DAB389(Gly4Ser)2-αMSH重组蛋白的构建、表达及纯化

Construction,expression and purification of recombinant protein DAB_(389)(Gly_4Ser)_2-αMSH with His-tag

目的:构建含有亲和标记His-tag的DAB389(Gly4Ser)2-αMSH重组蛋白,便于融合蛋白质的纯化。方法:利用含有His-tag序列、Factor Xa酶切识别序列、白喉毒素N端序列的基因作上游引物,含有αMSH全序列和(Gly4Ser)2互补序列基因作下游引物,以pET28a/DAB389(Gly4Ser)2EGF为模板,PCR扩增目的基因片段,并插入到原核表达载体pET28a中,构建了重组表达载体pET28a/亲和标记DAB389(Gly4Ser)2-αMSH,融合蛋白在BL21(λDE3)中以可溶形式表达,通过硫酸铵盐析、亲和层析纯化、脱盐、Factor Xa酶切得到重组蛋白纯品。结果:扩增的片段与理论值一致,序列分析正确;目的蛋白表达量约占菌体总蛋白量的30.6%;纯化得到纯度为94.36%的重组蛋白。结论:成功构建了含有His-tag的DAB389(Gly4Ser)2-αMSH重组蛋白,减少了纯化步骤,为进一步研究重组毒素、简便纯化途径奠定基础。

Objective： To construct the recombinant protein DAB389 （Gly4 Ser）2-αMSH containing His-tag in order to simplify purification. Methods： Gene fragment was amplified from plasmid pET28a/DAB389 （Gly4Ser）2-EGF by PCR with the upper primer containing His-tag, factor Xa distinguish sequence and diphtheria toxin complementary sequences, and the down primer containing αMSH and （ Gly4 Ser） 2 complementary sequences. The DAB389 （ Gly4 Ser） 2-αMSH gene fragment was amplified from plasmid pET28a/DAB389 （Gly4Ser）2-EGF by PCR, cloned into the expression vector pET28a （ ＋ ）, resuiting in plasmid pET28a/ DAB389 （GlyaSer）2-αMSH with His-tag. Recombinant plasmid was transformed into E. coli BL21 （ λDE3 ）and the recombinant toxin was expressed in a soluble protein. The protein was precipitated by ammonium sulfate, purified by Cu Chelating Sepharose Fast Flow, Superdex G-25 chromatography, then factor Xa was used for cleavage, the pure and DAB389 （Gly4Ser）2-αMSH was obtained. Results： The gene fragment was amplified right, the recombinant protein DAB389 （Gly4Ser）2-αMSH was constructed,expressed and accounted for 30.6% of the total protein. The purity of DAB389 （Gly4Ser） 2-αMSH was about 94.36%. Conclusion ： The recombinant protein DAB389 （Gly4Ser） 2-αMSH containing His-tag was successfully constructed, and the steps of purification were reduced which provides the basis for further research on the recombinant toxin and the predigest approaches.