García-Fruitós Elena, Carrió M Mar, Arís Anna, Villaverde Antonio
Institut de Biotecnologia i de Biomedicina and Departament de Genètica i de Microbiologia, Universitat Autònoma de Barcelona, Bellaterra, 08193 Barcelona, Spain.
Biotechnol Bioeng. 2005 Jun 30;90(7):869-75. doi: 10.1002/bit.20496.
In absence of chaperone DnaK, bacterially produced misfolding-prone proteins aggregate into large inclusion bodies, but still a significant part of these polypeptides remains in the soluble cell fraction. The functional analysis of the model beta-galactosidase fusion protein VP1LAC produced in DnaK(-) cells has revealed that the soluble version exhibits important folding defects and that it is less stable and less active than when produced in wild-type DnaK(+) cells. In addition, we have observed that the induction of gene expression at the very late exponential phase enhances twofold the stability of VP1LAC, a fact that in DnaK(-) background results in a dramatic increase of its specific activity up to phenotypically detectable levels. These results indicate that the chaperone DnaK is critical for the folding of misfolding-prone proteins and also that the soluble form reached in its absence by a fraction of polypeptides is not necessarily supportive of biological activity. In the case of E. coli beta-galactosidase, the catalytic activity requires assembling into tetramers and the fine organization of the activating interfaces holding the active sites, what might not be properly reached in absence of DnaK.
在缺乏伴侣蛋白DnaK的情况下，细菌产生的易于错误折叠的蛋白质会聚集形成大的包涵体，但这些多肽中仍有相当一部分保留在可溶性细胞组分中。对在DnaK(-)细胞中产生的模型β-半乳糖苷酶融合蛋白VP1LAC进行的功能分析表明，可溶性形式表现出重要的折叠缺陷，并且与在野生型DnaK(+)细胞中产生时相比，其稳定性更低且活性更弱。此外，我们观察到在指数生长后期诱导基因表达可使VP1LAC的稳定性提高两倍，在DnaK(-)背景下，这一事实导致其比活性急剧增加，直至达到表型可检测水平。这些结果表明，伴侣蛋白DnaK对于易于错误折叠的蛋白质的折叠至关重要，而且在其缺失的情况下一部分多肽所达到的可溶性形式不一定支持生物活性。就大肠杆菌β-半乳糖苷酶而言，催化活性需要组装成四聚体以及对保持活性位点的激活界面进行精细组织，而在没有DnaK的情况下可能无法正确实现这一点。
