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

Molecular Cloning, Expression and Purification of G-CSF Isoform D, an Alternative Splice Variant of Human G-CSF. Iran J Allergy Asthma Immunol. 2019 Aug 17;18(4):419-426 Authors: Toghraie FS, Yazdanpanah-Samani M, Mahmoudi Maymand E, Hosseini A, Asgari A, Ramezani A, Ghaderi A 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. ...
Source: Iranian Journal of Allergy, Asthma and Immunology - Category: Allergy & Immunology Authors: Tags: Iran J Allergy Asthma Immunol Source Type: research