厚壳贻贝转凝蛋白类贝壳基质蛋白的重组表达与功能分析

Recombinant expression and functional analysis of transgelin-like protein from the shell of Mytilus coruscus

为探明从厚壳贻贝(Mytilus coruscus)贝壳肌棱柱层中鉴定的一种新型贝壳基质蛋白——转凝蛋白类蛋白(transgelin-like protein,TLP)在贝壳形成过程中可能的分子机制,在序列分析基础上,对厚壳贻贝TLP进行原核重组表达及其表达产物的纯化,并分析其对方解石型和文石型碳酸钙晶体形貌的诱导作用及对晶型的影响,同时,分析其对2种晶型碳酸钙晶体结晶速度的抑制及结合作用。序列分析结果显示,厚壳贻贝TLP含有一段钙调理蛋白同源(calponin homology,CH)结构域,其三级结构以α-螺旋结构域为主。功能分析结果表明,重组厚壳贻贝TLP能诱导方解石型和文石型碳酸钙晶体在形貌上产生变化,其中:对方解石型碳酸钙晶体的晶型具有向文石型转化的作用,但对文石型碳酸钙晶体的晶型无影响。重组TLP对方解石型碳酸钙晶体的结晶速度具有明显的抑制作用,而对文石型碳酸钙晶体的结晶速度在蛋白质量浓度较高时具有促进作用。此外,重组TLP具有结合方解石型碳酸钙晶体的作用,但与文石型碳酸钙晶体无明显结合。以上结果表明,TLP对贝壳的形成具有影响并可能在贝壳肌棱柱层的形成中起到了重要作用。

Transgelin-like protein(TLP)is a novel shell matrix protein identified previously from the myostracum layer of Mytilus coruscus shell.For exploring its function in the shell formation of mussel,the TLP was recombinantly expressed by Escherichia coli expression system based on the sequence analysis and codon optimization.The functions of recombinant TLP(rTLP)on calcite-and aragonite-type calcium carbonate crystals were then investigated,including morphology,polymorph,crystallization rate,and binding ability of calcium carbonate crystals.Sequence analysis showed that the TLP contained a calponin homology(CH)domain,and the spatial structure of TLP predicted by SWISS-MODEL presented a conformation formed predominately byα-helices.Functional analyses showed that the rTLP had significant effects on the morphological change of aragonite-type calcium carbonate crystal and polymorph change of calcite-type calcium carbonate crystal,suggesting the function of this protein in the transformation of calcite to aragonite.In addition,the rTLP showed inhibition of calcite-type calcium carbonate crystallization rate in vitro,and promotion of aragonite-type calcium carbonate crystallization rate under high protein concentration.Moreover,the rTLP presented binding abilities to the calcite-type calcium carbonate crystal rather than the aragonite-type calcium carbonate crystal.Considering the myostracum layer is composed of aragonite-type calcium carbonate crystal,we speculate that the TLP may play important roles in the shell biomineralization and the formation of myostracum layer.