molecular cloning, expression and purification of g-csf isoform d, an alternative splice variant of human g-csf

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.