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

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，并将其与基因过度表达的策略相结合，以提高异源表达的性能。两种真核蛋白，即工业酶PalB和治疗性蛋白hCD83ext将被用作研究的模型分子。PalB含有三个分子内二硫键，对其溶解性和酶活性至关重要，而hCD83ext含有五个半胱氨酸残基，参与形成与其治疗生物活性相关的分子间和分子内二硫键。我们最近的实验结果基于这两种蛋白在大肠杆菌中的异源表达，表明了与体内二硫键形成相关的技术重要性，这可以在培养或下游加工的生物处理阶段以更有效、更严格、更特异和更可控的方式进行调节。为了克服这些技术障碍，作为拟议研究的目标，将对宿主和载体系统进行基因操作，以提高重组蛋白的表达水平、折叠效率、二硫键形成、稳定性和生物活性。科技发展主要包括：(1)用于基因过表达的重组DNA技术，(2)用于增加蛋白质溶解性的融合蛋白和伴侣共表达技术，(3)用于有效形成二硫键和精确定位不同表达间隔中的重组蛋白质的遗传系统的嫁接，(4)用于一致和可控的二硫键形成的突变衍生物的设计，以及(5)与这些用于生物活性开发和蛋白质回收的分子策略相关的下游生物处理。这项研究的结果不仅有助于提高对外源基因在大肠杆菌中过度表达的各种翻译后处理的效率，而且还将弥补目前限制真核蛋白在原核系统中表达的技术缺陷，用于许多生物制造应用。

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.