Ramping Recombinant Protein Expression in Bacteria.
Ramping Recombinant Protein Expression in Bacteria.
复制标题
提高细菌中重组蛋白的表达。
DOI：
10.1021/acs.biochem.0c00411
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发表时间：
2020
期刊：
影响因子：
2.9
通讯作者：
Gopalan,Venkat
中科院分区：
文献类型：
--
作者：
Zahurancik,WalterJ;Szkoda,BlakeE;Lai,LienB;Gopalan,Venkat
大肠杆菌是生物化学家对异源蛋白过表达的青睐的主力。尽管在大肠杆菌中高水平表达外源蛋白的方法看似简单，但表达不良和错误折叠是削弱重组蛋白生产的两个常见障碍。解决这些问题对于实现蛋白质的结构功能研究和扩大生物治疗的努力至关重要。在这里，我们强调了一项进展1，可以用于可预测地提高体内和体外的蛋白质表达。起始率和早期延伸率是翻译的起搏器，由多种因素决定，包括mRNA二级结构、tRNA丰度和密码子使用。越来越多的证据表明，这个列表应该包括开始密码子下游的核苷酸序列。例如，Voges等人2使用756个绿色荧光蛋白（GFP）融合构建物强调了该因子的重要性，每个构建物从第二个密码子开始具有不同的39-bp序列，以反映其在自然发生的开放阅读框（ORF）中的各自天然背景。他们观察到GFP表达与GC含量呈反相关，并预测碱基配对的可能性，特别是在密码子2和7之间。Goodman等人3从137个大肠杆菌必需基因的前33 bp中提取了14234个超级文件夹GFP基因，每个基因具有不同的启动子组合、核糖体结合位点和n端序列，得出了类似的结论。最后，Han等人4利用核糖体分析证实，在几种哺乳动物细胞系中，翻译orf的核糖体在密码子5处暂停，这一发现在他们对酵母、蠕虫和斑马鱼的核糖序列数据的再分析中得到了进一步的证实。因此，翻译效率（即单位时间内每个mRNA产生的全长多肽的数量）受到orf2,3 + 10附近的核苷酸和密码子5周围表示延伸承诺的强制性检查点的影响。4
Escherichia coli is the favored workhorse of biochemists for heterologous protein overexpression. Despite the deceptively straightforward approaches for expressing foreign proteins at high levels in E. coli, poor expression and misfolding are two common roadblocks that cripple production of recombinant proteins. Tackling these issues is essential to enable structurefunction studies of proteins and scale-up efforts of biotherapeutics. Here, we highlight an advance 1 that could be leveraged for predictably ramping protein expression in vivo and in vitro.The rates of initiation and early elongation, the pacemakers of translation, are determined by various factors, including mRNA secondary structure, tRNA abundance, and codon usage. There is a growing body of evidence that this list should include the nucleotide sequence immediately downstream from the start codon. For example, Voges et al. 2 highlighted the importance of this factor using 756 green fluorescent protein (GFP) fusion constructs, each with a different 39-bp sequence starting at the second codon to mirror its respective native context in a naturally occurring open reading frame (ORF). They observed that GFP expression anticorrelated with the GC content and predicted base-pairing likelihood, especially between codons 2 and 7. Goodman et al. 3 reached somewhat similar conclusions using their library of 14,234 superfolder GFP genes, each with a different combination of promoters, ribosome binding sites, and N-terminal sequences that were taken from the first 33 bp of 137 E. coli essential genes. Finally, Han et al. 4 used ribosome profiling to demonstrate ribosomal pausing at codon 5 of translating ORFs in several mammalian cell lines, a finding further cemented during their reanalysis of yeast, worm, and zebrafish Ribo-Seq data. Therefore, translation efficiency (ie, number of full-length polypeptides produced per mRNA per unit time) is influenced by the nucleotides around+ 10 of an ORF 2, 3 and by an obligatory checkpoint around codon 5 that signals elongation commitment. 4