马槟榔甜蛋白基因(MabinlinⅡ)的密码子优化及其在大肠杆菌中的表达

Codon Optimization and Expression of MabinlinⅡ Gene in Escherichia coli

本研究根据大肠杆菌密码子的偏好性,对马槟榔甜蛋白基因MabinlinⅡ(A链+B链)的密码子进行优化,并将优化后的马槟榔甜蛋白基因命名为MabinlinⅡ(AB)Plus。将MabinlinⅡ(AB)Plus基因与大肠杆菌表达载体pET-28a(+)片段连接,构建重组表达载体pET-28a(+)-MabinlinⅡ(AB)Plus,进而转化到大肠杆菌宿主表达菌株BL21(DE3),IPTG诱导蛋白表达。结果表明,密码子优化后的基因MabinlinⅡ(AB)Plus在大肠杆菌中最佳表达条件为:最适IPTG浓度1.4 mmol/L,最佳诱导剂温度为37℃,最适菌株起始生长量OD600为0.7,最佳诱导时间8 h。在最佳诱导表达条件下,目的蛋白表达量占菌体总蛋白约33.0%,与优化前约有提高7%。目的蛋白的上清液经过亲和纯化,在19 kDa处有单一的目的条带。经过感官评定,目的蛋白的甜度约为2%标准蔗糖溶液的400倍。这为研发以马槟榔甜蛋白为基础的新型甜味剂提供了科学的理论依据。

In this study,the codons of MabinlinⅡ(chainA+chainB)were optimized according to the codon usage bias in E.coli.The new sweet protein gene was named MabinlinII(AB)Plus.The MabinlinⅡ(AB)Plus gene was cloned into the E.coli expression vector pET-28a(+).The recombinant vector pET-28a(+)-MabinlinⅡ(AB)Plus was then transferred into E.coli BL21(DE3)host cells.Induction by IPTG,the optimized gene MabinlinⅡ(AB)Plus could be highly expressed in E.coli.The results showed that the optimal expression condition of the codon-optimized gene MabinlinⅡ(AB)Plus in E.coli was:IPTG concentration 1.4 mmol/L,inducer temperature was 37℃,optimum strain initial growth amount OD600,induction time was 0.7 h.Under the optimal induction expression conditions,the expression of the target protein expressed for about 33.0%of the total protein,which was about 7%higher than that before optimization.The supernatant of the target protein was affinity purified and had a single band at 19 kDa.After sensory evaluation,the sweetness of the target protein was about 400 times that of the 2%standard sucrose solution.This study would provide a scientific theoretical basis for the development of a new sweetener based on the sweet protein MabinlinⅡ.