厚壳贻贝足丝黏附蛋白mfp-3的重组表达及黏附功能分析

厚壳贻贝（Mytilus coruscus）中富含各种黏附蛋白分子，其中贻贝足丝蛋白3（mussel foot protein-3, mfp-3）是贻贝用以与外界基质进行黏附的主要蛋白分子.贻贝足丝中天然的mfp-3的含量低，水溶性差，因此纯化困难.本文以厚壳贻贝足丝蛋白mfp-3的cDNA序列为目的基因，用PCR法扩增Mfp-3基因,并成功构建含有多聚组氨酸标签的重组mfp-3原核表达载体pET-21a/ Mfp-3.经IPTG（isopropylthio-β-D-galactoside）诱导表达出重组蛋白，利用亲和层析和反相高效液相色谱分离纯化，获得分子量为9.18 kD的重组蛋白.经酪氨酸酶催化、玻璃包被和石英晶体微天平（quartz crystal microbalance，QCM）分析.结果表明,重组厚壳贻贝mfp-3蛋白经酪氨酸酶催化后,L-3,4-二羟基苯丙氨酸(即多巴,L-3,4- dihydroxyphenylalanine, DOPA) 含量较高并且具有较好的黏附性能.上述研究为开发以mfp-3黏附蛋白为来源的生物粘合剂奠定了良好的基础.

Recombinant Expression and Functional Analysis of Mytilus coruscus Foot Protein-3

Mussels Mytilus coruscus can adhere to various solid surfaces in the presence of moisture. Mussel foot protein-3 (mfp-3) has been suggested as the main adhesive protein in the plaques closest to the adhesion interface and also has been suggested as potential environmentally friendly adhesives for use in aqueous conditions and in medicine. To obtain enough M. coruscus mfp-3 for the further study, using E. coli expression system, recombinant mfp3 with a 6×His tag at C-termini was successfully expressed and purified by Ni-NTA affinity purification followed by reverse phase HPLC separation. The actual molecular mass of recombinant mfp3 was determined as 9.18 kD by MALDI-TOF, which is almost identical to the calculated mass of 9.15 kD and confirmed that recombinant mfp-3 had been successfully purified to a high level. Recombinant mfp3 was modified by tyrosinase and DOPA content assay was performed to detect the conversion efficiency of Tyr to DOPA. Adhesion ability tests such as coating assay and QCM revealed that recombinant mfp3 has significant adhesion ability especially after modified by tyrosinase. These assays revealed the correlation between DOPA content and adhesion ability, and that recombinant mfp3 has significant adhesion ability thus may be useful as a bioadhesive in medical or underwater environments.