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

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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.