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相互作用，以介导细胞环境中的正确折叠，以及（2）操纵这些途径，以实现具有生物活性的异源蛋白质的成本效益生产。 其具体目标是：（1）获得关于由clpB转录物合成的替代翻译产物的体内功能的机制信息，因为它们已经显示ClpB在从头蛋白质折叠中起重要作用，这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，他们将调查E。大肠杆菌需要ClpB（或ClpB变体）与DnaK-DnaJ-GrpE的精确化学计量比以促进聚集倾向蛋白的正确折叠。(2)使用易错PCR诱变产生ClpB变体，并筛选所得文库中表现出增强的伴侣蛋白功能的突变体。选择的功能突变体的增益将被纯化和表征，以获得对ClpB的结构-功能关系的见解。有用的氨基酸取代将在单个基因上组合，并且将评估各种突变体改善易聚集蛋白质折叠的能力。(3)确定是否可以通过将新合成的多肽的通量重定向到DnaK-DnaJ-GrpE团队及其辅助分子伴侣来改善小的聚集倾向蛋白的折叠。这将通过消除染色体上的触发因子（tig）基因来实现。同时，他们将使用遗传学方法分离E。组成型合成高水平的主要分子伴侣组的大肠杆菌菌株，并将在这些背景中引入TIG缺失，目的是产生可能特别适合于产生可溶形式的异源蛋白质的宿主细胞。将在分批和补料分批发酵中评估这些菌株的性能。 预期所提出的工作将产生有用的工具来处理异源蛋白质聚集的问题，同时为ClpB的功能和作用机制以及伴侣蛋白辅助蛋白质在E.杆菌

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.