Chaperone Assisted Protein Folding in E. coli

PI的长期目标是：(1)了解分子伴侣和折叠酶如何与蛋白质底物相互作用，以及如何在细胞环境中介导适当的折叠；(2)操纵这些途径以实现具有生物活性的异源蛋白的成本效益生产。他们的具体目标是：(1)获得由clpB转录物合成的替代翻译产物在体内功能的机制信息，因为他们已经表明clpB在蛋白质从头折叠中起重要作用，可能是由于其独特的分解和重塑蛋白质聚集体的能力。作为这一目标的一部分，他们将研究大肠杆菌是否需要精确的ClpB（或ClpB变体）与dna - dnaj - grpe的化学计量比来促进易于聚集的蛋白质的正确折叠。(2)利用易出错的PCR诱变技术生成ClpB变体，并筛选具有增强伴侣蛋白功能的突变体文库。选择的功能突变体将被纯化和表征，以深入了解ClpB的结构-功能关系。有用的氨基酸替换将被组合在一个基因上，并且各种突变体改善顽固聚集倾向蛋白折叠的能力将被评估。(3)通过将新合成多肽的通量重新定向到dak - dnaj - grpe团队及其附属伴侣，确定是否可以改善小聚集易感蛋白的折叠。这将通过从染色体上消除触发因子（tig）基因来实现。与此同时，他们将使用遗传学方法分离大肠杆菌菌株，这些菌株组成性地合成高水平的主要伴侣蛋白组，并将在这些背景中引入tig缺失，目的是产生特别适合以可溶性形式产生异种蛋白的宿主细胞。这些菌株的性能将在分批发酵和补料分批发酵中进行评估。预计本研究将为解决异源蛋白聚集问题提供有用的工具，同时为了解ClpB的功能和作用机制以及大肠杆菌细胞质中伴侣辅助蛋白折叠的途径提供基础见解。

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.