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Translational regulation of periplasmic folding assistants and proteases as a valuable strategy to improve production of translocated recombinant proteins in Escherichia coli.
BMC Biotechnology ( IF 3.5 ) Pub Date : 2020-05-11 , DOI: 10.1186/s12896-020-00615-0
Agnieszka Gawin 1 , Helga Ertesvåg 1 , Sine Alise Hartvigsen Hansen 1 , Jostein Malmo 2 , Trygve Brautaset 1
Affiliation  

BACKGROUND Advantages of translocation of recombinant proteins to the periplasm in Escherichia coli include simplified downstream processing, and improved folding and in vivo activity of the target protein. There are, however, problems encountered in the periplasmic production that can be associated with the incorrect formation of disulfide bonds, incomplete cleavage of the signal peptide, and proteolytic degradation. A common strategy used to overcome these difficulties involves manipulating the cellular levels of proteases and periplasmic folding assistants like chaperones, signal peptide peptidases or thiol-disulfide oxidoreductases. To date, this has been achieved by plasmid-based over-expression or knockouts of the relevant genes. RESULTS We changed the translation efficiencies of five native E. coli proteins, DsbA, DsbB, Skp, SppA, and DegP, by modifying the strength of their ribosome binding sites (RBS). The genomic RBS sequences were replaced with synthetic ones that provided a predicted translation initiation rate. Single- and double-gene mutant strains were created and tested for production of two pharmaceutically relevant proteins, PelB-scFv173-2-5-AP and OmpA-GM-CSF. Almost all the single-gene mutant strains showed improved periplasmic production of at least one of the recombinant proteins. No further positive effects were observed when the mutations were combined. CONCLUSIONS Our findings confirm that our strain engineering approach involving translational regulation of endogenous proteins, in addition to plasmid-based methods, can be used to manipulate the cellular levels of periplasmic folding assistants and proteases to improve the yields of translocated recombinant proteins. The positive effects of SppA overexpression should be further investigated in E. coli.

中文翻译:

胞质折叠助剂和蛋白酶的翻译调控是提高大肠杆菌中易位重组蛋白产量的重要策略。

背景技术重组蛋白易位至大肠杆菌周质的优势包括简化的下游加工,以及靶蛋白的折叠和体内活性的改善。然而,在周质生产中遇到的问题可能与二硫键的不正确形成,信号肽的不完全切割以及蛋白水解降解有关。用于克服这些困难的常见策略涉及操纵蛋白酶和周质折叠助剂(如伴侣蛋白,信号肽肽酶或巯基-二硫键氧化还原酶)的细胞水平。迄今为止,这已经通过基于质粒的相关基因的过表达或敲除来实现。结果我们改变了5种天然大肠杆菌蛋白DsbA,DsbB,Skp,SppA和DegP的翻译效率,通过修改其核糖体结合位点(RBS)的强度。基因组RBS序列被合成的RBS序列替代,该序列提供了预期的翻译起始速率。创建了单基因和双基因突变株,并测试了两种药物相关蛋白(PelB-scFv173-2-5-AP和OmpA-GM-CSF)的产生。几乎所有的单基因突变株都显示出至少一种重组蛋白的周质产生得到改善。当组合突变时,没有观察到进一步的积极作用。结论我们的发现证实,除了基于质粒的方法外,我们的菌株工程方法还涉及内源蛋白的翻译调控,可用于操纵周质折叠助剂和蛋白酶的细胞水平,以提高易位重组蛋白的产量。SppA过表达的积极作用应在大肠杆菌中进一步研究。
更新日期:2020-05-11
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