Cloning, expression, purification and characterization of human hematopoietic growth factor expressed in E.coli.

Author(s): Aruna Adusumilli*, Ramakrishna Rao K., Sambasiva Rao K.R.S., Madhavi Chandra

Abstract

One of the human Hematopoietic growth factor GCSF, which effects the proliferation, differentiation and activation of hematopoietic progenitor cells, expressed in E. coli. The main objective of this study was to clone, express, purified and characterize rh-GCSF. Since GCSF expressed in E. coli is a non-glycosylated and has been potentially used for treating neutropenia in Oncology and Hematology. The source of this product is Bladder carcinoma cell line, reverse transcribed, cloned and expressed in E. coli 1, we have used an efficient procedure for purification of rh-GCSF from E. coli since most of the eukaryotic proteins in E. coli is expressed as insoluble aggregates as inclusion bodies 2, were homogenized, properly washed for the clearance of impurities, contaminants, host cell proteins etc., solubilized in chaotropic agents and refolded for proper three dimensional conformation of the protein. Final purification with Ion exchange chromatography for the removal of trace of impurities, nucleic acids, host cell and proteins. Purity was measured by SDS-PAGE. Final protein yields were 0.6 gm. /liter. Further characterization studies like molecular weight determination by SDS-PAGE and MALDI, N-terminal sequencing and sensitivity and specificity conformation by Western blotting results have showed promising results and it is more pure, free of endotoxins (LAL test) and ready to use for preclinical studies.

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