Molecular Cloning, Expression and Purification of GCSF Isoform D, an Alternative Splice Variant of Human GCSF

Granulocyte colonystimulating factor (GCSF) is the major regulator of hemopoiesis and granulopoiesis. However, overexpression of GCSF 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 GCSF (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 GCSF transcript variant 4 encoding GCSF isoform D from a highly malignant tumor cell line (Mehr80) with overexpression of GCSF. Both the fulllength GCSF isoform B and GCSF isoform D were cloned from Mehr80 cell line, overexpressed in Escherichia coli as Nterminal glutathioneStransferase fusion proteins in the form of inclusion bodies and affinity purified by the batch method using glutathioneSepharose 4B resin. Both fusion proteins were successfully cloned and expressed. Folded recombinant proteins were solubilized from inclusion bodies using sarkosyl, Triton X114 and CHAPS and purified. The purity of GCSF isoforms was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and they were clearly detected in western blot analysis using antiGCSF polyclonal antibody. The GCSF plays various roles in physiological and pathological conditions, however to date, the differential function of GCSF isoforms remains unknown. Considering the fact that GCSF isoform D was isolated from a highly malignant tumor cell line with overexpression of GCSF, the role of this splice variant in tumorigenesis requires further investigation.