Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm
Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm
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DOI：
10.1073/pnas.96.24.13703
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发表时间：
1999-11-23
影响因子：
11.1
通讯作者：
Georgiou, G
中科院分区：
文献类型：
--
作者：
Bessette, PH;Åslund, F;Georgiou, G
在生理条件下，大肠杆菌细胞质保持在还原状态，这强烈不利于蛋白质中稳定二硫键的形成。然而，其中硫氧还蛋白和谷胱甘肽的还原受损的突变体（trxB戈尔突变体）在细胞质中积累氧化的、酶活性的碱性磷酸酶。这些突变体在缺乏外源还原剂的情况下生长非常差，并以高频率积累基因外抑制子。一种这样的抑制菌株，FA113，生长几乎一样快的野生型在还原剂的情况下，表现出略快的二硫键形成的动力学，并具有完全诱导的转录激活因子，OxyR的活性。与野生型或trxB突变株相比，FA113得到了显著更高的适当氧化蛋白质产量。对于具有非常复杂的二硫键模式的多肽，如vtPA和全长tPA，通过共表达具有不同氧化还原电位的TrxA（硫氧还蛋白1）突变体，活性蛋白的量进一步提高至15倍，或通过蛋白质二硫键异构酶DsbC提高至20倍。值得注意的是，通过在E. coli FA 113中，与通过分泌到野生型菌株的周质中可以实现的相比，即使在优化的条件下。这些结果表明，可以使细胞质充分氧化，以允许有效形成天然二硫键，而不损害细胞活力。
Under physiological conditions, the Escherichia coli cytoplasm is maintained in a reduced state that strongly disfavors the formation of stable disulfide bonds in proteins. However, mutants in which the reduction of both thioredoxins and glutathione is impaired (trxB gor mutants) accumulate oxidized, enzymatically active alkaline phosphatase in the cytoplasm. These mutants grow very poorly in the absence of an exogenous reductant and accumulate extragenic suppressors at a high frequency. One such suppressor strain, FA113, grows almost as rapidly as the wild type in the absence of reductant, exhibits slightly faster kinetics of disulfide bond formation, and has fully induced activity of the transcriptional activator, OxyR. FA113 gave substantially higher yields of properly oxidized proteins compared with wild-type or trxB mutant strains. For polypeptides with very complex patterns of disulfide bonds, such as vtPA and the full-length tPA, the amount of active protein was further enhanced up to 15-fold by coexpression of TrxA (thioredoxin 1) mutants with different redox potentials, or 20-fold by the protein disulfide isomerase, DsbC. Remarkably, higher yields of oxidized, biologically active proteins were obtained by expression in the cytoplasm of E. coli FA113 compared with what could be achieved via secretion into the periplasm of a wild-type strain, even under optimized conditions. These results demonstrate that the cytoplasm can be rendered sufficiently oxidizing to allow efficient formation of native disulfide bonds without compromising cell viability.