Recombinant Pharmaceutical Protein from E. coli

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A wide variety of recombinant protein expression methods have been developed for pharmaceutical and structural studies. E.coli expression system has been most widely used to produce sufficient amount of heterologous proteins, and the development of its new technologies is still continuing.

In general, post-translational modifications of eukaryotic proteins such as glycosylation and phosphorylation are absent when the protein is recombinantly expressed in prokaryotic host cells. In many cases where it is required that the heterologous protein form complex disulfide bonds, its overexpression in soluble form in the cytoplasm of bacterial host cells is difficult since the cytoplasm is an unsuitable environment for disulfide bond formation due to the highly reduced environment. However, the overexpression of recombinant heterologous protein is generally achieved using bacterial expression systems, and especially those employing the Gram-negative bacterial strain E. coli, since prokaryotic host cells offer many practical advantages: easy genetic manipulation, simple cultivation handling, no requirement of special culture rooms or specialized equipment, rapid cell growth and large amount of biomass, resilience and adaptability to a wide range of culture conditions and the availability of costeffective sophisticated 2H/13C/15N stable isotope-labeling technology. Consequently, approximately 30% of recombinant therapeutic products on the market in recent times are manufactured in E. coli. Use of an E. coli expression system is the first choice when the molecular weight of the desired protein is < 100 kDa and any post-translational modifications of the target protein are not taken to be indispensable for proper structure formation and biological activity. The most commonly used technology for recombinant protein expression using living E. coli host cells is the pET vector system. There are several important steps to pay more attention when employing the E. coli strains, such as template mRNA sequence optimization, solubility-enhancement tags fused to the terminal end of the heterologous protein, structural biological analysis of peptides, etc.

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Figure 1. Schematic representations of pET and pCold vector expression systems of E. coli.[1]

Reference
[1] T. Sugiki et al. (2014) Latest approaches for efficient protein production in drug discovery. Expert Opin. Drug Discov. P(1189-1204).

For research use only.

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