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微生物内进行蛋白质表达前需要考虑的8件事

发布: 2016-01-31 1,514阅读 216评论

在微生物内进行蛋白质表达是生物科技中的核心技术。然而，每种蛋白质都具有其独特的结构性质，比如二硫键（disulfide bonds）数量，是否存在疏水区域 (hydrophobic regions)，是否存在跨膜区域 (transmembrane domains)，是否具有糖基化 (glycosylations) 等翻译后修饰过程等， 所有的这些性质都可能决定着蛋白质表达过程的难易。在与目的蛋白质来源相似的宿主内表达蛋白质要相对容易些。然而，大多时候，我们需要在与蛋白质来源不同的宿主内进行蛋白质的表达，比如在原核细胞内表达真核生物的蛋白质，这通常是具有挑战性的。在这里，小编与大家分享几个有助于跨系统实现蛋白质表达的工具。

1密码子优化

为了理解密码子优化(codon optimization)的重要性，我们需要首先知道什么是密码子优化。简单来讲，就是在我们构建的载体内引入宿主系统tRNA更愿意读取的密码子，这样能够更容易将其翻译成氨基酸，这会提高翻译速度，提高蛋白质表达的效率。没错，我这里用了“更愿意”这样一个主观性很强的词。实际中确实是这样的，生物系统是具有密码子偏好性的，不论真核生物还是原核生物，每种生物都会表现出某种程度的密码子利用的差异和偏爱。这是因为不同生物系统对于特定的密码子具有不同数量的可利用的tRNAs，并且每一个系统内对于每一个密码子，都有特定数量的tRNAs。所以，如果tRNAs与密码子数量的比例不正确，就会阻碍蛋白质的表达。因此，在进行蛋白质表达前，需要根据表达系统，对目的基因进行密码子优化，使用宿主系统内存在的tRNAs更偏好的密码子。

2纯化标签

当我们在微生物内进行蛋白质的表达时，最后通常都是要对蛋白质进行纯化的。当然，如果你只是为了应用全细胞提取物或者裂解提取物，可以不用考虑蛋白质的纯化问题。为了实现蛋白质的纯化，我们必须为蛋白质加上纯化标签。目前很多纯化标签已经被系统研究并应用于实践了，常用的纯化标签包括His tag， Strep-II tag， GST， Flat-Tag等。有关蛋白质的纯化标签，大家可以点击链接，查看BioEngX历史文章。

3分子伴侣与折叠酶

表达一个困难的基因就像砸碎一个硬坚果，有时候你需要借助外力。为了获得一个有用的蛋白，很重要的一点就是保证蛋白质正确折叠。细胞质（cytoplasm）的环境还原性(reducing)的，这不利于二硫键的正确形成，因此会影响蛋白质的折叠。为了辅助蛋白质正确的折叠，防止形成不可溶的包涵体，我们需要表达分子伴侣(chaperones)和折叠酶(foldases)的辅助载体。一些常见的分子伴侣和折叠酶包括DsbA/C, Skp, FkpA, GroEL/ES, DnaK/J/GrpE等。我们可以根据蛋白质的表达位置，选择细胞质（cytoplasmic）内分子伴侣或者细胞间质（periplasmic）分子伴侣。另外一些标签也能够辅助蛋白质的折叠，比如麦芽糖结合蛋白（maltose binding protein, MBP）。MBP是一个42.5 KDa的蛋白质，能够与目的蛋白质融合表达，不仅能够增加蛋白质的可溶性，还能够辅助蛋白质折叠。MBP的另一个优势是能够与交联淀粉柱（amylose column）结合实现目标蛋白的纯化。

4蛋白质表达位置

在实验之前，我们需要考虑蛋白质在哪里表达，是细胞质内（cytoplasm），细胞间质（periplasm）还是细胞外(extracellular)分泌表达。如果一个蛋白质内包含有二硫键，我们最好在细胞间质的氧化环境内令其表达。氧化环境能够帮助蛋白质形成正确的二硫键从而获得正确的结构。如果想要蛋白质在细胞内特定的位置表达或分泌到细胞外，需在蛋白质的N端添加信号肽（signal sequence）。在信号肽的帮助下，蛋白质能够转移到特定的位置。例如，来自果胶杆菌（Pectobacterium carotovorum）内的PelB信号肽能够引导蛋白质进入到革兰氏阴性菌（gram-negative）的细胞间质中。再比如，来自变铅青链霉菌（Streptomyces lividans）内的信号肽能够将使蛋白质分泌表达到培养基之中。

5蛋白质表达通路

蛋白质分泌表达具有很多的优势。一方面分泌表达蛋白质能够减少对宿主菌的毒性和代谢负担，使菌适应性增加；另一方面周质空间和胞外培养基中宿主菌蛋白含量很低，有利于目的蛋白的纯化。不同的表达途径对蛋白质的折叠效率有很大的影响。革兰氏阴性菌共分为5种天然分泌系统，即I型，II型，III型，IV型和V型。 E.coli 主要通过I型和II型系统分泌表达蛋白质。Ⅰ型分泌系统通过α-溶血素途径直接介导胞外分泌，组成元件简单，外源重组蛋白与HlyAC-端结合后，再与HlyB/HlyD形成复合物，由ATP水解提供能量，通过TolC通路实现胞外分泌。通过该系统分泌出胞外的蛋白C端仍然带有一段信号序列，需要在体外进行剪切;此外该系统中的一些共表达部件对目的蛋白的转运有竞争作用，重组蛋白表达量很低。Ⅱ型分泌系统先介导细胞周质分泌，再经过MTB(main terminal branch)机制实现胞外分泌。分泌蛋白利用以下三种途径透过细胞质膜: Sec (secretion)通路、信号识别颗粒(signal recognition particle, SRP)通路或双精氨酸转移(twin arginine translocation, TAT)通路。三种通路有所不同，Sec通路将蛋白质转移到周质空间后蛋白质进行折叠，TAT通路却是将折叠后的蛋白质转移到细胞周质，而SRP途径允许蛋白质同时进行折叠与转移的过程[1]。由于Ecoli跨越内膜的转运装置不完善，蛋白输出能力不够,蛋白酶降解等原因导致分泌效率低，胞外分泌量往往达不到实验或工业生产的要求，目前大肠杆菌的分泌主要是指重组蛋白通过Ⅰ或Ⅱ型系统透过胞质膜分泌至周质腔[2]。

6表达菌株

不同的大肠杆菌菌株在培养条件和外源基因表达能力上存在着很大差别，因此不同宿主菌的选择对表达产物的积累以及下游分离纯化有至关重要的作用。利用蛋白酶表达缺陷的突变菌株可以提高重组蛋白的分泌表达量,一些缺陷性菌株(如合成外膜元件的基因突变体)既能够产生可溶性的、具有正确空间结构和二硫键的活性蛋白，又能够避免细胞壁在外源蛋白跨膜转运中的阻碍作用，有利于重组蛋白分泌到培养基中。最成功的例子就是L2型细菌(缺乏细胞壁和胞周质)已用于青霉素酰基转移酶、链激酶、微小抗体的分泌表达中。但因为这些菌株有生长受损性，不能耐受高密度发酵的过程，L2型细菌不适合工业化生产。总之有很多种类的菌株可以用来实现蛋白质的表达。有关大肠杆菌的菌株及表达载体，大家可以从这个网站了解到，Ecoliwiki.net。

7培养基，温度，诱导条件等因素

培养基成分、培养方式、培养条件及培养过程中抑制性代谢产物的积累等都会影响重组蛋白在工程菌中的表达产量和分泌的量。其中培养基营养成分、pH和温度等参数是影响工程菌生长和表达外源蛋白的重要因素,能够影响蛋白酶的活性、分泌和表达水平。向培养基中添加甘氨酸可以在不导致菌体自溶的情况下促进蛋白从周质空间向胞外的分泌,且不引起明显的细菌裂解。在培养基中添加糖胶和TritonX-100能阻止周间腔中包涵体的形成，并提高胞外表达的效率。改变培养基的渗透压、短时间的热冲击诱导及降低诱导时的温度,会明显提高重组蛋白在大肠杆菌的可溶性表达。另外，对于需要IPTG诱导的菌株，诱导时刻，温度及培养时间必须经过优化。

8细胞培养时间

合适的细胞培养时间是决定着细胞完整度及蛋白质表达水平的一个重要因素。细胞培养时间过长会导致细胞裂解，目的蛋白丢失，有毒蛋白酶释放。而细胞培养时间太短的直接后果就是细胞浓度不够，目标蛋白产率太低。因此重组菌株的培养时间必须经过优化。

Reference:

【1】Valent QA, Scotti PA and et al. The Escherichia coli SRP and SecB targeting pathways converge at the translocon. EMBO J. 1998 May 1;17(9):2504-12.

【2】郑海洲,刘晓志,宋欣.重组蛋白在大肠杆菌分泌表达的研究进展.天津药学杂志,2009,21（4）：0040-42.

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