Molecular and bioprocessing strategies in posttranslational processing to enhance recombinant protein production

翻译后加工中提高重组蛋白产量的分子和生物加工策略

• 批准号: 283237-2009
• 负责人: Chou, CPerry
• 金额: $ 1.68万
• 依托单位: University of Waterloo
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2013
• 资助国家: 加拿大
• 起止时间: 2013-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=525644
• 关键词: Molecular; bioprocessing; strategies; posttranslational; processing

In this proposed research, various molecular and bioprocessing aspects associated with disulfide bond formation will be explored and integrated with the strategies for gene overexpression to improve the performance of heterologous expression. Two eukaryotic proteins, i.e. an industrial enzyme of PalB and a therapeutic protein of hCD83ext, will be used as the model molecules for study. PalB contains three intramolecular disulfide bonds which are critical for its solubility and enzymatic activity, whereas hCD83ext contains five cysteine residues involved in the formation of intermolecular and intramolecular disulfide bonds related with its therapeutic bioactivity. Our recent experimental results based upon the heterologous expression of these two proteins in E. coli suggest the technical importance associated with in vivo disulfide bond formation, which could be mediated in a more effective, rigorous, specific, and controllable manner at the bioprocessing stage of either cultivation or downstream processing. To overcome these technical hurdles as the objective of the proposed research, the host and vector system will be genetically manipulated to enhance the expression level, folding efficiency, disulfide bond formation, stability, and bioactivity of the recombinant proteins. The scientific and technical development primarily includes: (1) recombinant DNA technology for gene overexpression, (2) fusion protein and chaperone coexpression technologies to increase the protein solubility, (3) graft of a genetic system for effective formation of disulfide bonds and precise targeting of recombinant proteins in various expression compartments, (4) design of mutant derivatives for consistent and controllable disulfide bond formation, and (5) downstream bioprocessing associated with these molecular strategies for bioactivity development and protein recovery. The outcome of the proposed research is expected to not only shed light on enhancing the efficiency of various posttranslational processings upon heterologous gene overexpression in E. coli but also complement the technical deficiencies currently limiting the expression of eukaryotic proteins in the prokaryotic system of E. coli for many biomanufacturing applications.

在这项拟议的研究中，将探索与二硫键形成相关的各种分子和生物处理方面，并与基因过表达的策略进行探索，以改善异源表达的性能。两种真核蛋白，即PALB的工业酶和HCD83EXT的治疗蛋白，将用作研究模型分子。 PALB包含三种分子内二硫键，这对于其溶解度和酶活性至关重要，而HCD83EXT ext含有五个参与与其治疗生物活性相关的分子间和分子内二硫化物键的涉及的半胱氨酸残基。我们最近基于大肠杆菌中这两种蛋白质的异源表达的实验结果表明，与体内二硫键形成相关的技术重要性，可以在培养或下游加工的生物过程中以更有效，严格，特定，可控制的方式介导。为了克服这些技术障碍作为拟议研究的目标，将在遗传上操纵宿主和向量系统，以提高重组蛋白的表达水平，折叠效率，二硫键键形成，稳定性和生物活性。科学和技术发展主要包括：（1）重组DNA技术用于基因过表达，（2）融合蛋白和伴侣蛋白共表达技术，以提高蛋白质溶解度，（3）遗传系统的移植物，用于有效形成二硫键的遗传系统，并在各种表达式中的精确靶向构造，以构成各种表达型的构造（4）均匀的构造（4）突变的构造（4）突变的（4）突变的（4）突变的（4）突变的构造，（4）突变的（4）突变的构造（4）。 （5）与这些分子策略有关生物活性发展和蛋白质恢复的下游生物处理。预计拟议的研究的结果不仅阐明了大肠杆菌中异源基因过表达的各种翻译后过程的效率，而且还补充了当前限制了许多生物制造应用原核系统中真核蛋白的技术缺陷。