蜘蛛丝蛋白基因的合成及其串联体在大肠杆菌中的表达  

Synthesis of Spider Silk-protein Gene and Expression of its Multimers in Escherichia coli

作　　者：周培[1] 邹竹荣[1] 曹丽娟[1] 苏裕[1] 

机构地区：[1]华东师范大学生命科学学院,上海200062

出　　处：《中国农业科技导报》2007年第6期71-77,共7页Journal of Agricultural Science and Technology

基　　金：上海市科委纳米科技专项(05nm05016)资助

摘　　要：赋有许多独特性能的天然蛋白质纤维——蜘蛛丝在生物医学、材料和军事等领域具有广泛的应用前景，但惟有通过基因工程手段才能大量获取。根据已报道的蜘蛛丝蛋白的氨基酸序列，结合蜘蛛丝蛋白的模块结构特性和功能的关系，优化设计并人工合成了全长为429bp的蜘蛛丝蛋白基因SPS。将该基因克隆到质粒pET-32a中，构建成表达载体pET-SPS1，并进一步构建了它的多个串联体表达载体pET—SPS（2～8）。所有表达载体在大肠杆菌BL21（DE3）中进行了诱导表达，重组蜘蛛丝蛋白得到了纯化。结果表明，合成的蜘蛛丝蛋白基因与预期一致，融合表达的蜘蛛丝蛋白几乎全部可溶。其中，1—3串联体蜘蛛丝蛋白基因能够有效表达，但随着串联数的进一步增加，蜘蛛丝蛋白基因融合表达效率下降，初步探讨了下降的可能原因。Spider silks, being a class of nature protein fibers with many functional properties, have large potentials in the fields of biomedical, material and military applications, which are however to be sufficiently obtained through the only pathway of genetic engineering. In this study, an artificial spider silk-protein gene, SPS of the full length of 429bp, was optimally designed and synthesized on the basis of the previously reported amino acid sequences of several spider silk proteins, in which the features of structural modules and their functional correlations were particularly concerned. This synthetic gene was sub-cloned in plasmid pET-32a to generate the expression vector pET-SPS1 which was further used as the backbone for creation of a set of expression vectors, pET-SPS （2 ~ 8 ） having different muhimers of SPS gene. Finally, all SPS vectors were expressed in E. coli strain BL21 （DE3） by IPTG induction, and the recombinant SPS fusion proteins were purified. From our results, the synthetic spider silk protein gene SPS was verified identical to the design. All SPS fusion proteins expressed in E. coli are almost fully soluble. The vectors with I - 3 multimers of SPS could be efficiently expressed, while those of more increased multimers could only give rise to poor expression. A possible interpretation for this scenario was proposed.

关 键 词：蜘蛛丝蛋白 基因合成 串联体 蛋白质表达 大肠杆菌 

分 类 号：Q78[生物学—分子生物学]