一种新型贝壳基质蛋白的重组表达与功能分析

Recombinant expression and functional analysis of a new type of shell matrix protein

贝壳是在贝壳基质蛋白的调控下形成的生物矿化产物,也是生物工程和生物材料学研究的重要对象。贝壳基质蛋白通过调控碳酸钙晶体的成核和生长过程,使贝壳形成有规则的纳米结构并赋予贝壳极强的力学性能。在厚壳贻贝肌棱柱层中鉴定到一种新型贝壳基质蛋白,因其富含谷氨酰胺而被命名为富含谷氨酰胺的贝壳蛋白(Glutamine-rich shell protein,GRSP),该蛋白序列中含18.2%的谷氨酰胺以及PDZ(Postsynaptic density/Discs large/Zonula occludens)和ZM(ZASP-like Motif)结构域。为了解GRSP在生物矿化过程中的分子机理,在序列分析的基础上,采用密码子优化结合原核重组表达策略,获得重组厚壳贻贝GRSP;进一步利用扫描电镜和傅里叶红外光谱,分析重组GRSP对方解石型以及文石型碳酸钙晶体在形貌和晶型方面的影响;同时,利用沉淀法分析重组GRSP对碳酸钙结晶速度的影响以及与碳酸钙晶体的结合作用。结果表明,重组GRSP可诱导文石型碳酸钙晶体发生形貌变化,并对方解石型碳酸钙晶型产生影响。此外,重组GRSP表现出与碳酸钙结合的活性并能抑制方解石型碳酸钙晶体的结晶速度。上述结果表明,GRSP蛋白可能对贝壳的形成及发育具有重要影响并在贝壳肌棱柱层的形成中发挥重要作用。

It is recognized that mollusk shell is composed mainly by calcium carbonate crystal and produced via mineralization process tightly controlled by shell matrix proteins,as a prominent material for biomaterials and bioengineering study.Shell matrix proteins play an important role in the formation of shell,as well as the regulation of calcium carbonate biomineralization and the promotion of shell growth.A novel Glutamine-rich shell protein(GRSP)with 18.2%Gln has been identified from the myostracum layer of Mytilus coruscus shell previously.A PDZ(postsynaptic density/discs large/zonula occludes)domain and a ZM(ZASP-like motif)domain have been detected in the sequence of GRSP.However,the roles of GRSP in shell formation are still unclear and no related research has been reported yet.For understanding the mechanism of the GRSP in biomineralization,the GRSP of M.coruscus is recombinant expressed by E.coli BL21 strain with codon optimization.The function of recombinant GRSP is studied and the results showed that,recombinant GRSP can affect the crystal morphology of aragonite calcium carbonate,as well as the polymorph of calcite calcium carbonate.Furthermore,the recombinant GRSP can interact with the calcium carbonate crystal and inhibit the crystalline growth rate of calcite.These results suggest that GRSP may be a biomineralizationrelated protein in the shell of M.coruscus and play an important role in the formation of the myostracum layer.