High level expression of recombinant human growth hormone in Escherichia coli: crucial role of translation initiation region

Abstract

For high-throughput production of recombinant protein in Escherichia coli (E. coli), besides important parameters such as efficient vector with strong promoter and compatible host, other important issues including codon usage, rare codons, and GC content specially at N-terminal region should be considered. In the current study, the effect of decreasing the percentage of GC nucleotides and optimizing codon usage at N-terminal region of human growth hormone (hGH) cDNA on the level of its expression in E. coli were investigated. Mutation in cDNA of hGH was performed through site-directed mutagenesis using PCR. Then, the mutant genes were amplified and cloned into the expression vector, pET-28a. The new constructs were transformed into the BL21(DE3) strain of E. coli and chemically induced for hGH expression. At the final stage, expressed proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), scanning gel densitometry, and western blot. SDS-PAGE scanning gel densitometry assay and western blot analysis revealed higher expression level of hGH by using the two new expressions constructs (mutant genes vectors with decreasing GC content and optimized-codon usage at N-terminal of cDNA) in comparison with wild gene expression vector. Obtained results demonstrated that decreasing the GC nucleotide content and optimization of codon usage at N-terminal of the hGH cDNA could significantly enhance the expression of the target protein in E. coli. Our results highlight the important role of both 5´ region of the heterologous genes in terms of codon usage and also GC content on non-host protein expression in E. coli.

Keywords: Codon usage; Escherichia coli; GC content; Recombinant human growth hormone; pET-28a.

Fig. 1

Mutant N-terminally modified hGH cDNA amplification by PCR. (M) 1kb DNA ladder; (lane 1) intact cDNA; (lane 2) N-terminally modified cDNA with codon optimization; (lane 3) N-terminally modified cDNA with GC content reduction; and (lane 4) blank sample.

Fig. 2

Gel electrophoresis of PCR colony reaction of transformants. (lane 1) transformant with pET-rhGH1; (lane 2) transformant with pET-rhGH2 (N-terminally modified cDNA with codon optimization); (lane 3) transformant with pET- rhGH3 (N-terminally modified cDNA with GC content reduction); and (M) 1Kb marker.

Fig. 3

Gel electrophoretic analysis of double digestion reactions of (lane 2) pET-rhGH1; (lane 3) pET-rhGH2; and (lane 4) pET-rhGH3 with XhoI-NheI enzymes. Lanes 5, 6, 7 illustrate the non-digested pET-rhGH1, pET-rhGH2, and pET-rhGH3 constructs, respectively. Lane 1 demonstrates the 1kb DNA ladder.

Fig. 4

SDS-PAGE analysis of expressed rhGH. (Lane 1) induced BL21(DE3) containing pET-rhGH2 construct; (lane 2) induced BL21(DE3) containing pET-rhGH3 construct; (lane 3) induced BL21(DE3) containing pET-rhGH1 construct; and (M) protein ladder.

Fig. 5

Western blot analysis of rhGH. (Lane 1) BL21(DE3) cell lysate as negative control; (lane 2) induced BL21(DE3) containing pET-rhGH2 construct; (lane 3) induced BL21(DE3) containing pET-rhGH3 construct; (lane 4) induced BL21(DE3) containing pET-rhGH1 construct; and (lane 5) standard rhGH.