Renner Julie N, Kim Yeji, Cherry Kevin M, Liu Julie C
School of Chemical Engineering, Purdue University, West Lafayette, IN 47907-2100, USA.
Protein Expr Purif. 2012 Mar;82(1):90-6. doi: 10.1016/j.pep.2011.11.019. Epub 2011 Dec 8.
Resilin has emerged as a promising new biomaterial possessing attractive properties for tissue engineering applications. To date, proteins with repeating resilin motifs have been expressed with molecular weights less than 30 kDa. This work describes the development of resilin-based proteins (repeating motif derived from Anopheles gambiae) 50 kDa in size. A modular cloning scheme was utilized and features a recursive cloning technique that can seamlessly and precisely tune the number of resilin repeats. Previously-established resilin expression protocols (based on the Studier auto-induction method) were employed to express the proteins in Escherichia coli BL21(DE3)pLysS. Western blot and densitometry results demonstrated that only ~50% of expressed proteins were the desired molecular weight. This finding suggested that either protein truncation or degradation occurred during protein expression. Preventing leaky expression, lowering the culture temperature, and harvesting during exponential phase resulted in up to 94% of the expressed proteins having the desired molecular weight. These expression conditions differ from previously-published resilin expression methods and are recommended when expressing proteins with a larger number of repetitive resilin sequences.
弹性蛋白已成为一种有前景的新型生物材料，具有适用于组织工程应用的诱人特性。迄今为止，具有重复弹性蛋白基序的蛋白质表达分子量小于30 kDa。这项工作描述了大小为50 kDa的基于弹性蛋白的蛋白质（重复基序源自冈比亚按蚊）的开发。采用了模块化克隆方案，其特点是一种递归克隆技术，可无缝且精确地调整弹性蛋白重复序列的数量。使用先前建立的弹性蛋白表达方案（基于Studier自诱导方法）在大肠杆菌BL21(DE3)pLysS中表达蛋白质。蛋白质印迹和密度测定结果表明，只有约50%的表达蛋白是所需的分子量。这一发现表明，在蛋白质表达过程中发生了蛋白质截短或降解。防止渗漏表达、降低培养温度并在指数生长期收获，使得高达94%的表达蛋白具有所需的分子量。这些表达条件与先前发表的弹性蛋白表达方法不同，在表达具有大量重复弹性蛋白序列的蛋白质时推荐使用。
