Natural Sciences - Research article Open Access Logo

Cloning, periplasmic expression and purification of hGM-CSF (Human Granulocyte - Macrophage ColonyStimulating Factor) in Escherichia coli

Phu Sang Nguyen 1, *
Thanh Thao Nguyen 1
Hieu Tran Van 1
  1. University of Science, VNU-HCM
Correspondence to: Phu Sang Nguyen, University of Science, VNU-HCM. Email: pvphuc@vnuhcm.edu.vn.
Volume & Issue: Vol. 17 No. 4 (2014) | Page No.: 12-19 | DOI: 10.32508/stdj.v17i4.1551
Published: 2014-12-31

Online metrics


Statistics from the website

  • Abstract Views: 2230
  • Galley Views: 1223

Statistics from Dimensions

Copyright The Author(s) 2023. This article is published with open access by Vietnam National University, Ho Chi Minh city, Vietnam. This article is distributed under the terms of the Creative Commons Attribution License (CC-BY 4.0) which permits any use, distribution, and reproduction in any medium, provided the original author(s) and the source are credited. 

Abstract

Human GM-CSF is a cytokine consisting of 127 amino acid residues, with four cysteines being involved in two disulfide bonds. Although GM-CSF is glycosylated in its natural form, the glycosylation perhaps has not been involved in its biological function. GM-CSF stimulates the survival, proliferation, and differentiation of hem at opoietic progenitor cells and also enhances the functional properties of mature myeloid cells. GM-CSF is used as a therapeutic agent in various clinical cases such as neutropenia following chemotherapy, bone marrow transplantation, acute myeloid leukemia… In this study, we report the results on the cloning and expression of recombinant human GM-CSF in the periplasmic space of Escherichia coli. The hGM-CSF gene was amplified by polymerase chain reaction using two oligonucleotide primers containing NcoI and XhoI restriction sites. This DNA fragment was successfully cloned between the NcoI and Xho I sites of the plasmid pET-22b, in frame with the pelB signal peptide sequence. The expression vector pET-hGM was transformed into E. coli BL21(DE3) and the transformants were induced by IPTG and examined for hGM-CSF production. Periplasmic proteins were released by osmotic shock treatment. The expression of recombinant hGM-CSF was evaluated by SDS–PAGE in total, cytoplasmic and periplasmic fractions. The recombinant hGMCSF in periplasmic fraction was then subjected to ion exchange chromatography using Q Sepharose FF column with saltincrement elution step. SDS-PAGE showed there was a visible expression of recombinant hGM-CSF in the periplasmic fraction of the E. coli BL21(DE3)/pET-hGM and a purified band with the purity of 97.4% after ion exchange chromatography. This result was further confirmed by Western blot using anti-hGM-CSF antibody.

Comments