Molecular Cloning, Expression and Purification of G-CSF Isoform D, an Alternative Splice Variant of Human G-CSF

  • Fatemeh Sadat Toghraie Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
  • Mahsa Yazdanpanah-Samani Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
  • Elham Mahmoudi Maymand Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
  • Ahmad Hosseini Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
  • Amir Asgari Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
  • Amin Ramezani Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran AND Department of Medical Biotechnology, School of Advanced Medical Sciences and Technologies, Shiraz University of Medical Sciences, Shiraz, Iran
  • Abbas Ghaderi Shiraz Institute for Cancer Research, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran AND Department of Immunology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran
Keywords: Alternative splicing, Glutathione-S-transferase, Granulocyte colony-stimulating factor, Leukocytosis, Malignant tumors, Recombinant fusion protein

Abstract

Granulocyte colony-stimulating factor (G-CSF) is the major regulator of hemopoiesis and granulopoiesis. However, overexpression of G-CSF has been implicated in several important processes in tumor biology such as tumor growth, angiogenesis, and metastasis. Four different mRNA isoforms resulting from alternative splicing have been reported for G-CSF (transcript variants 1, 2, 3 and 4). The mRNAs and protein products of splice variants 1 and 2 have been isolated for the first time, from tumor cell lines. In the present study for the first time we isolated the G-CSF transcript variant 4 encoding G-CSF isoform D from a highly malignant tumor cell line (Mehr80) with overexpression of G-CSF. Both the full-length G-CSF isoform B and G-CSF isoform D were cloned from Mehr80 cell line, overexpressed in Escherichia coli as N-terminal glutathione-S-transferase fusion proteins in the form of inclusion bodies and affinity purified by the batch method using glutathione-Sepharose 4B resin. Both fusion proteins were successfully cloned and expressed. Folded recombinant proteins were solubilized from inclusion bodies using sarkosyl, Triton X-114 and CHAPS and purified. The purity of G-CSF isoforms was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and they were clearly detected in western blot analysis using anti-G-CSF polyclonal antibody. The G-CSF plays various roles in physiological and pathological conditions, however to date, the differential function of G-CSF isoforms remains unknown. Considering the fact that G-CSF isoform D was isolated from a highly malignant tumor cell line with overexpression of G-CSF, the role of this splice variant in tumorigenesis requires further investigation.

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Published
2019-08-17
How to Cite
1.
Toghraie F, Yazdanpanah-Samani M, Mahmoudi Maymand E, Hosseini A, Asgari A, Ramezani A, Ghaderi A. Molecular Cloning, Expression and Purification of G-CSF Isoform D, an Alternative Splice Variant of Human G-CSF. Iran J Allergy Asthma Immunol. 18(4):419-426.
Section
Original Article(s)