重组Maxadilan的制备与鉴定

Preparation and Identification of Recombinant Maxadilan

摘要:

为利用基因工程技术获得重组Maxadilan(RMMAX), 根据Maxadilan的氨基酸序列, 设计并人工合成了在原核表达的基因。克隆到表达载体pKYB, 重组质粒pKYB-MAX转化表达宿主菌Escherichia coli strain ER2566, 构建表达工程菌。用诱导剂IPTG诱导由目的多肽、内含肽和几丁质结合域(Chitin binding domain, CBD)组成的“三元”融合蛋白表达; 用几丁质珠亲和层析纯化了裂解液中的融合蛋白, 用b-巯基乙醇切割融合蛋白, 获得目的蛋白。所得的多肽经激光飞行质谱测定分子量结果与理论值相符, 生物活性分析表明, 重组Maxadilan有显著的提升血糖的作用。

Abstract:

To produce recombinant Maxadilan using gene engineering technology, the gene of recombinant Maxadilan which expressed in protocaryon were designed and synthesized according to the amino acid sequences of Maxadilan. The recombinant plasmid pKYB-MAX was constructed and transformed into host bacteria Escherichia coli strain ER2566. After the MAX-intein-CBD fusion protein was purified by chintin-affinity chromatography, the self-cleavage activity of the intein was induced by β-mercaptoethanol and the recombinant Maxadilan was released from the chitin-bound intein tag. The molecular weight of peptides was determined by the laser flight mass spectrometry and the results was conformity with the theoretical value. The biological activity analysis showed that recombinant Maxadilan significantly enhanced the concentrat ion of serum glucose.