蜘蛛丝蛋白基因的合成及其串联体在大肠杆菌中的表达.pdfVIP

中国农业科技导报，2007，9(6)：71—77 Journal of Agricultural Science and Technology 蜘蛛丝蛋白基因的合成及其串联体在大肠杆菌中的表达 周 培， 邹竹荣， 曹丽娟， 苏 裕 (华东师范大学生命科学学院，上海200062) 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，优化没计并人工合成了全长为429 bp的蜘蛛丝蛋白基因SPS。将该基因克隆到质 粒pET一32a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。 关键词：蜘蛛丝蛋白；基因合成；串联体；蛋白质表达；大肠杆菌 中图分类号：Q78 文献标识码：A 文章编号：1008-0864(2007)06-0071-07 Synthesis of Spider Silk-protein Gene and Expression of its Multimers in Escherichia coli ZHOU Pei，Z0U Zhu—Rong，CAO Li—Juan，SU Yu (School ofLife Science，East China Normal University，Shanghai 200062，China) Abstract：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一32a 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 difierent muhimers of SPS gene．Finally，all SPS vectors were expressed in E coli strain BL21(DE3)by IPTG induction，and the recombi