Proteome profiling and its application in developing recombinant escherichia coli strains

Abstract

Proteome profiling of microorganisms have been carried out to generate valuable knowledge that can be used for the development of metabolic and cellular engineering strategies, which consequently are used to enhance the yield and productivity of native or foreign bioproducts and to modify cellular properties to improve mid-stream and down-stream processes. For these tasks, the proteome profiling by two-dimensional gel electrophoresis technology combined with mass spectrometry have been applied to the development of the strategies for the metabolic and cellular engineering of Escherichia coli and other applications. First, proteome analysis was carried out to understand metabolic and physiological changes of E. coli during the high cell density cultivation (HCDC). It provided invaluable information in designing metabolic engineering and fermentation strategies for the production of recombinant proteins and metabolites by HCDC of E. coli. Second, cellular and metabolic perturbation of recombinant E. coil during fed-batch culture for the production of human leptin was understood by proteomes analysis. Based on proteome profiles, highly depressed CysK enzyme during human leptin production was identified and engineered, which finally lead to improved growth of recombinant E. coli and three- to four-fold increase in the productivity of serine-rich recombinant proteins. Third, proteome profiling of the inclusion body (IB) fraction of recombinant protein produced in E. coil suggested that small heat shock proteins, IbpA and IbpB, are major proteins bound to the lBs. They are essential and play important roles in the production of recombinant proteins in E. coli by protecting recombinant proteins from the degradation by proteases. Based on these findings, two possible applications of IbpA and IbpB in the production of recombinant proteins were demonstrated: (1) enhanced production of recombinant proteins by overexpressing the ibpA and/or ibpB genes and (2) enhanced secr...