在大肠杆菌中高效表达外源蛋白的策略(3)
2007-11-15 21:52:42   来源：本站原创   评论：0 点击：

培养条件
     E.coli中的蛋白质产量可以通过高细胞密度培养系统而获得显著提高。高细胞密度培养系统可以分成三类：分批培养、补料分批培养和连续培养。这些方法能获得超过100g/升的细胞浓度，从而获得廉价的重组蛋白。有关大规模培养系统的资料已有详细的综述[193，209]。培养基的组成需要仔细地计算和监控，因为这对细胞和蛋白质的产生具有重要的代谢效应。例如，不同mRNA的翻译可以因温度和培养基的变化而受到不同程度的影响[210]。营养成分和培养参数如pH、温度和其他参数都会影响蛋白酶的活性、分泌和产量[211]。已经证明对培养基的特殊操作能明显提高蛋白质释放到培养基中。例如，在培养基中添加甘氨酸能增强外周质蛋白释放到培养基中，且不引起明显的细菌裂解[212，213]。同样，在山梨糖醇和甘氨酰甜菜碱存在的渗透压力下培养细菌，可以使可溶性的活性蛋白产量提高多达400倍[139]。
高细胞密度培养系统也有其自身的缺陷。这些缺陷包括在高细胞密度情况下，溶解氧的量有限；二氧化碳水平能够降低生长速度、刺激乙酸形成降低发酵罐的混合效率和产热等。有关这些问题的解决方案已经有详细的介绍[193]。利用高细胞密度培养系统生产重组蛋白质的一个主要问题是乙酸的积累，这种亲脂性成分对细胞的生长是有害的[193]。虽然有多种解决这一问题的方案，但是均各有缺点。近来这一问题通过将来自B. subtilis编码醋酸盐合成酶的alsS基因导入E.coli细胞中得以解决[141]。该酶催化丙酮酸转化为非酸性和低毒性的副产品。乙酸积累的减少极大地改善了重组蛋白的产生。另外，其他酶缺陷的E.coli突变株也已经建立，这些突变株产生较少的乙酸，从而提高了人重组蛋白的表达水平[214]。
 
总结
一种高效的原核表达载体需要包括一个强大并且可以严紧调节的启动子；一位于翻译起始密码子5’端大约9bp的SD序列；位于目的基因3’末端的一个高效转录终止子。除此之外，载体还需要一个复制起点，筛选标记和利于对启动子活性进行严紧调节的基因。这种调节元件可以插入载体自身，也可以插入宿主的染色体。其他的元件包括转录和翻译增强子等。这些元件的作用往往具有基因特异性，因此要根据不同的情况加以取舍。我们在综合国内外研究的基础上，对有可能影响E.coli中蛋白质高效表达的多种因素进行了总结。尽管目前在E.coli中表达外源基因方面已经有许多重大进展，但仍有许多问题亟待解决。归纳起来主要有以下几点：
1．  借助于细胞的分子伴侣机制来提高正确折叠的蛋白质的产量。或许可以通过共表达多种分子伴侣编码基因和通过激细胞内众多不同的分子伴侣的方法来实现。
2．在E.coli中表达真核膜蛋白和多亚基蛋白质复合物的难题上有待解决。
3．  蛋白质分泌到培养基中的真正和有效机制需要明了。目前已经有多种体系能够是重组蛋白质分泌到培养基中。其中有些是利用信号肽、融合伴侣和具有穿透能力的因子，这种因子能够引起外膜的破坏和有限的渗漏。而另外的策略是利用已经存在的分泌通道，来保证更高特异性的分泌。有关这方面的工作需要对E.coli中众多分泌通道的更多了解。
4．  赋予原核细胞诸如真核细胞那样进行翻译后修饰的能力。例如糖基化、磷酸化、乙酰化和酰氨化等。可以通过将合适的真核细胞酶类转入E.coli中而达到这些目的。
总之，E.coli作为一种原核表达系统具有多方面的优点，加之在许多技术方面的众多重大进展。E.coli仍然是基础研究和商业生产重组蛋白质的强有力工具。

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