Comparison of T7- and Lac-Based Systems for the Periplasmic Expression of Human Granulocyte Macrophage Colony Stimulating Factor in Escherichia coli

Document Type: Research Paper

Authors

National Research Center for Genetic Engineering and Biotechnology, P.O. Box: 14155-6343, Tehran, Iran.

Abstract

With the aim of the production of recombinant human granulocyte macrophage colony stimulating factor  (rhGM-CSF) in the periplasmic space of Escherichia coli, the expression of hGM-CSF cDNA was examined
under the regulation of a T7-based as well as a lac-based expression systems. For the efficient expression of hGM-CSF cDNA, the first five codons at the N-terminal were altered based on the E. coli major codon usage. The hGM-CSF cDNA, fused to pelB signal sequence, was expressed using the two inducible promoters. The expression analysis of the 2 recombinant plasmids were performed in the BL21(DE3) and TG1 strains of E. coli, respectively. After induction with 1mM isopropyl-ß-DThiogalactopyranoside (IPTG) the recombinant E.
coli with T7 promoter produced hGM-CSF more efficiently than did the lac promoter. Under inducing conditions
both of the recombinant bacteria allowed successful secretion of hGM-CSF into the periplasmic space. The optimal temperature for the over-expression of the recombinant protein under the T7-based system was 30°C and that of the lac regulated system was 28°C. The optimization of growth condition for the recombinant bacteria, produced in this work, provides mean for studying the function of environmental as well as genetic factors on the overexpression of recombinant proteins in the periplasmic space of E. coli.

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