重组贻贝黏蛋白Mgfp-5的表达及功能评价

The Expression and Function Evaluation of the Recombinated Mussel Adhesive Protein Mgfp-5

贻贝(Mytilus)中富含各种黏附蛋白,其中贻贝足丝蛋白5(Mytilus galloprovincialis foot protein type 5,Mgfp-5)是贻贝与外界基质进行黏附的主要蛋白分子之一。贻贝黏蛋白天然提取时产量低、易凝固且纯化困难,通过化学合成和基因工程等方法重组贻贝黏蛋白解决该问题。本研究在E.coli BL21(DE3)中表达了贻贝蛋白Mgfp-5,亲和层析纯化到Mgfp-5蛋白的含量为12.25%,纯度为96.92%;酪氨酸酶修饰Mgfp-5后,其DOPA(多巴,3,4-二羟基苯丙氨酸)含量为9.60 pmol/g,是修饰前的9.32倍,生物黏性增强到1 116 n N,是Cell-Tak TM黏性的1.60倍;3.0μg/cm2的Mgfp-5蛋白的毒性级别为1级;Mgfp-5浓度≤20μg/m L时对红细胞无溶血现象,具有生物安全性。本研究可为重组Mgfp-5蛋白研究奠定基础,同时为来源于Mgfp-5黏蛋白的临床生物黏合剂的研制提供参考。

Mytilus contains a variety of adhesive proteins, among which Mytilus galloprovincialis foot protein type 5（Mgfp-5） is one of the major proteins to stick with the substrates. Natural extraction of Mgfp-5 could obtain very little purified protein since the process is labor-intensive and inefficient. While the recombination of mussel adhesive protein through chemical synthesis and genetic engineering could overcome these limitations. In this research, the recombinant protein Mgfp-5 was expressed in E. coli BL21（DE3） and purified by affinity chromatography（12.25% production yield and 96.92% purity）. After modifying by tyrosinase, the DOPA content in Mgfp-5 was 9.60 pmol/g and the adhesion enhanced to 1 116 n N, which were 9.32 times and 1.6 times as large as unmodified Mgfp-5, respectively. The toxicity level of 3.0 μg/cm2 Mgfp-5 protein was of the first level. The hemolysis of red blood cells didn＇t appear when the concentration of Mgfp-5 protein was no more than 20 μg/m L,which meant that it was bio-safe. This study could provide the basis for the recombination of Mgfp-5 protein and provide reference for the development of clinical bioadhesive of Mgfp-5 as well.