一种新型贻贝足丝抗氧化蛋白的原核重组表达及功能

贻贝足丝及其足丝蛋白相关研究对于开发新型水下生物粘附剂具有重要的仿生学意义。足丝蛋白在其粘附过程中需要维持一定的还原态，而目前已报道的足丝抗氧化蛋白仅有MFP-6。此前在厚壳贻贝足丝中鉴定到一种新型的富含半胱氨酸和甘氨酸的足丝蛋白质，该蛋白质被命名为Cys/Gly-Rich-Protein（CGRP），但是CGRP蛋白在足丝中的作用及机制尚不明确。为此，针对CGRP蛋白，在序列分析基础上，利用原核重组表达手段获得其重组蛋白质，采用2,2-联苯基-1-苦基肼基（2,2-diphenyl-1-picryl hydrazyl radical，DPPH）法检测CGRP重组蛋白经不同条件处理后的抗氧化活性。序列分析结果表明，CGRP蛋白含16.5%的半胱氨酸和10%的甘氨酸，其序列中含有两段半胱氨酸位置保守的重复序列，结构预测表明，其优势构象以无规卷曲为主。同源蛋白质搜索结果表明，CGRP蛋白在数据库中尚无高同源性蛋白质存在。通过密码子优化结合原核重组表达策略成功表达出CGRP重组蛋白，所获得的CGRP重组蛋白具有明显的抗氧化活性，且该活性在其半胱氨酸还原后显著增强(0.91±0.05 vs 0.71±0.11, P<0.01)而在半胱氨酸烷基化之后显著下降(0.08±0.03 vs 0.71±0.11, P<0.01)，表明CGRP蛋白的抗氧化活性与其序列中半胱氨酸的自由巯基有关。本研究提示，CGRP蛋白是足丝中一种新的具有抗氧化功能的蛋白质，在足丝粘附过程中推测与MFP-6一起参与了富含多巴的足丝粘附蛋白的还原态维持，对贻贝足丝在固化和粘附过程中防止提前粘附具有重要意义。

Recombinant Expression and Functional Analysis ofa Novel Mussel Byssal Protein

The studies on mussel byssus and byssal proteins have significant importance for the development of novel underwater biological adhesives in biomimetics. The main components of mussel byssus are various adhesive proteins named mussel foot proteins (MFPs) and these proteins need to be maintained as reduction state during the adhesion process. For now, the only reported MFP with antioxidant function is MFP-6, a cycteine-rich protein. Except for the MFP-6, another novel MFP with abundant cysteine and glycine, named Cys/Gly-Rich-Protein (CGRP), was identified from Mitilus coruscus in previous work. However, the function and the role of CGRP in mussel byssus is unclear. Therefore, the sequential analysis was firstly performed for CGRP. The recombinant CGRP protein was obtained by prokaryotic recombinant expression system with codon optimization, and the antioxidant activity of recombinant CGRP was determined by the method of 2, 2-diphenyl 1-picryl hydrazyl radical (DPPH) after different treatments. The results of sequence analysis showed that CGRP protein contains 16.5% cysteine and 10% glycine. Two internal repeats, each one has 7 conserved cysteines, are presented in the sequence. In addition, the results of homologous searching revealed that there is no homologous of CGRP in protein database. Recombinant CGRP (rCGRP) fused with a hexa-histidine affinity tag was successfully expressed in Escherichia coli with 40 mg/L cell culture, and purified by affinity chromatography. Purified rCGRP has higher DPPH radical scavenging activity comparing to Vitamin C. The DPPH radical scavenging activity of rCGRP was enhanced significantly after dithiothreitol reduction (0.91±0.05 vs 0.71±0.11, P<0.01). On the other hand, the activity was significantly weakened after iodoacetamide modification (0.08±0.03 vs 0.71±0.11, P<0.01). These results indicated that the antioxidant activity of rCGRP is associated with the free cysteine sulfhydryl. This study suggests that CGRP protein is a novel antioxidant protein in the byssus. It is speculated that CGRP, together with MFP-6, is involved in the reduction maintenance of dopa-rich MFPs during the adhesion process, which is important to prevent mussel byssus from early adhesion before byssal solidification and adhesion.