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

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Toghraie Fatemeh Sadat; YAZDANPANAH SAMANI MAHSA; MAHMOUDI MAYMAND ELHAM; HOSSEINI AHMAD; ASGARI AMIR; RAMEZANI AMIN; GHADERI ABBAS

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Journal: Iranian Journal of Allergy, Asthma and Immunology Year:2019 | Volume:18 | Issue:4 Page(s): 419-426

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Title

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

Author(s)

Keywords

Alternative splicing

Glutathione-S-transferase

Granulocyte colonystimulating 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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APA: Copy

Toghraie, Fatemeh Sadat, YAZDANPANAH SAMANI, MAHSA, MAHMOUDI MAYMAND, ELHAM, HOSSEINI, AHMAD, ASGARI, AMIR, RAMEZANI, AMIN, & GHADERI, ABBAS. (2019). Molecular Cloning, Expression and Purification of G-CSF Isoform D, an Alternative Splice Variant of Human G-CSF. IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY (IJAAI), 18(4), 419-426. SID. https://sid.ir/paper/291637/en

Vancouver: Copy

Toghraie Fatemeh Sadat, YAZDANPANAH SAMANI MAHSA, MAHMOUDI MAYMAND ELHAM, HOSSEINI AHMAD, ASGARI AMIR, RAMEZANI AMIN, GHADERI ABBAS. Molecular Cloning, Expression and Purification of G-CSF Isoform D, an Alternative Splice Variant of Human G-CSF. IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY (IJAAI)[Internet]. 2019;18(4):419-426. Available from: https://sid.ir/paper/291637/en

IEEE: Copy

Fatemeh Sadat Toghraie, MAHSA YAZDANPANAH SAMANI, ELHAM MAHMOUDI MAYMAND, AHMAD HOSSEINI, AMIR ASGARI, AMIN RAMEZANI, and ABBAS GHADERI, “Molecular Cloning, Expression and Purification of G-CSF Isoform D, an Alternative Splice Variant of Human G-CSF,” IRANIAN JOURNAL OF ALLERGY, ASTHMA AND IMMUNOLOGY (IJAAI), vol. 18, no. 4, pp. 419–426, 2019, [Online]. Available: https://sid.ir/paper/291637/en

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