Chaperone Assisted Protein Folding in E. coli

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The PI's long term objective is to: (1) Understand how molecular chaperones and foldases interact with protein substrates and each other to mediate proper folding in the cellular environment, and (2) Manipulate these pathways to achieve cost-effective production of biologically active heterologous proteins. Their specific aims are to: (1) Gain mechanistic information on the in vivo function of the alternate translation products synthesized from the clpB transcript since they have shown that ClpB plays an important role in de novo protein folding, possibly as a result of its unique ability to break apart and remodel protein aggregates. As part of this aim, they will investigate whether E. coli requires a precise stoichiometric ratio of ClpB (or ClpB variants) to DnaK-DnaJ-GrpE to promote the correct folding of aggregation-prone proteins. (2) Use error-prone PCR mutagenesis to generate ClpB variants and screen the resulting libraries for mutants exhibiting enhanced chaperone function. Selected gain of function mutants will be purified and characterized to gain insights on the structure-function relationship of ClpB. Useful amino acid substitutions will be combined on a single gene, and the ability of various mutants to improve the folding of recalcitrant aggregation-prone proteins will be assessed. (3) Determine whether the folding of small aggregation-prone proteins can be improved by redirecting the flux of newly synthesized polypeptides to the DnaK-DnaJ-GrpE team and its ancillary chaperones. This will be accomplished by eliminating the trigger factor (tig) gene from the chromosome. In parallel, they will use genetics approaches to isolate E. coli strains that constitutively synthesize high levels of the major chaperone teams and will introduce the tig deletion in these backgrounds with the goal of generating host cells that may be particularly well suited for producing heterologous proteins in a soluble form. The performance of these strains will be assessed in batch and fed-batch fermentations. It is anticipated that the proposed work will generate useful tools to deal with the problem of heterologous protein aggregation while providing fundamental insights on the function and mechanism of action of ClpB and the pathways of chaperone-assisted protein folding in the cytoplasm of E. coli.