Research in Pharmaceutical Sciences

ORIGINAL ARTICLE
Year
: 2017  |  Volume : 12  |  Issue : 2  |  Page : 168--175

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


Mahsa Ghavim1, Khalil Abnous2, Fatemeh Arasteh3, Sahar Taghavi2, Maryam Sadat Nabavinia5, Mona Alibolandi2, Mohammad Ramezani4 
1 Damghan Branch, Islamic Azad University, Damghan, I.R. Iran
2 Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, I.R. Iran
3 Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, I.R. Iran
4 Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences; Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, I.R. Iran

Correspondence Address:
Mohammad Ramezani
Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences; Nanotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad
I.R. Iran
Mona Alibolandi
Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad
I.R. Iran

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.


How to cite this article:
Ghavim M, Abnous K, Arasteh F, Taghavi S, Nabavinia MS, Alibolandi M, Ramezani M. High level expression of recombinant human growth hormone in Escherichia coli: crucial role of translation initiation region.Res Pharma Sci 2017;12:168-175


How to cite this URL:
Ghavim M, Abnous K, Arasteh F, Taghavi S, Nabavinia MS, Alibolandi M, Ramezani M. High level expression of recombinant human growth hormone in Escherichia coli: crucial role of translation initiation region. Res Pharma Sci [serial online] 2017 [cited 2022 Jan 17 ];12:168-175
Available from: https://www.rpsjournal.net/article.asp?issn=1735-5362;year=2017;volume=12;issue=2;spage=168;epage=175;aulast=Ghavim;type=0