Co-production of GroELS discriminates between intrinsic and thermally-induced recombinant protein aggregation during substrate quality controlReportar como inadecuado




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Microbial Cell Factories

, 10:79

First Online: 12 October 2011Received: 05 September 2011Accepted: 12 October 2011

Abstract

BackgroundThe effects and effectiveness of the chaperone pair GroELS on the yield and quality of recombinant polypeptides produced in Escherichia coli are matter of controversy, as the reported activities of this complex are not always consistent and eventually indicate undesired side effects. The divergence in the reported data could be due, at least partially, to different experimental conditions in independent research approaches.

ResultsWe have then selected two structurally different model proteins namely GFP and E. coli β-galactosidase and two derived aggregation-prone fusions to explore, in a systematic way, the eventual effects of GroELS co-production on yield, solubility and conformational quality. Host cells were cultured at two alternative temperatures below the threshold at which thermal stress is expected to be triggered, to minimize the involvement of independent stress factors.

ConclusionsFrom the analysis of protein yield, solubility and biological activity of the four model proteins produced alone or along the chaperones, we conclude that GroELS impacts on yield and quality of aggregation-prone proteins with intrinsic determinants but not on thermally induced protein aggregation. No effective modifications of protein solubility have been observed, but significant stabilization of small encapsulable substrates and moderate chaperone-induced degradation of larger excluded polypeptides. These findings indicate that the activities of this chaperone pair in the context of actively producing recombinant bacteria discriminate between intrinsic and thermally-induced protein aggregation, and that the side effects of GroELS overproduction might be determined by substrate size.

Electronic supplementary materialThe online version of this article doi:10.1186-1475-2859-10-79 contains supplementary material, which is available to authorized users.

Gemma Platas, Escarlata Rodríguez-Carmona, Elena García-Fruitós contributed equally to this work.

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Autor: Gemma Platas - Escarlata Rodríguez-Carmona - Elena García-Fruitós - Olivia Cano-Garrido - Antonio Villaverde

Fuente: https://link.springer.com/



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