Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones
Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones
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DOI：
10.1379/csc-139r.1
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发表时间：
2005-12-01
影响因子：
3.8
通讯作者：
Goloubinoff, P
中科院分区：
文献类型：
--
作者：
de Marco, A;Vigh, L;Goloubinoff, P
当重组蛋白在细菌中大量表达时，往往会错误折叠并以可溶和不可溶的无功能聚集体的形式积累。改善重组蛋白质天然折叠的一般策略是增加粘性有机化合物(称为渗透压物质)或分子伴侣的细胞浓度，这些化合物可以防止聚集，并可以主动清除聚集并将聚集转化为自然可折叠的物种。在这项研究中，金属亲和纯化(固定化金属离子亲和层析[iMac])，证实了对胰酶消化的抵抗力，以区分可溶性聚集体和可溶性类天然蛋白。在诱导蛋白质合成的过程中，盐诱导的渗透调节物质的积累显著提高了折叠顽抗蛋白的IMAC产量。然而，共表达DNAK-DNAJ-GRPE、ClpB、GroEL-Groes和IbpA/B的细胞获得了最高的产量。将膜流变剂热休克诱导剂苯甲醇(BA)添加到细菌培养基中，获得了与质粒介导的伴侣蛋白共表达类似的高产量。我们的结果表明，简单的BA介导的内源伴侣的诱导可以取代要求更高的伴侣共表达的方法。为了研究和生物技术的目的，可以认为将渗透分子诱导的自然折叠与热、BA或血浆诱导的伴侣蛋白共表达相结合的策略可以优化细菌中自然折叠的重组蛋白的产量。
When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmidinduced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.