Abstract

An improved in vitro expression system called the rapid translation system (RTS) was used in this study for the in vitro biosynthesis of 6x His tagged granulocyte colony stimulating factor (6x His-tagged granulocyte colony-stimulating factor (GCSF)). This was done to overcome the problems associated with traditional cell based biotechnology. The study involved the preparation of template DNA for cell-free protein synthesis through gene amplification of open reading frame (ORF) of hGCSFb, cloning in pIVEX 2.4d vector and transformation of the produced construct in chemically competent Escherichia coli DH 5 α cells. A cell free protein synthesis system, RTS 100 E. coli HY kit, was tested for 6x His tagged G-CSF protein synthesis. Protein purification was done using Ni-NTA chromatography. Protein production was detected by two methods electrophoretically by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunologically by dot blotting immunodetection. The use of these methods yielded purified 6xHis-tagged GCSF with a concentration of about 250 µg/ml RTS reaction.

Key words: Granulocyte colony stimulating factor, in vitro expression system, RTS systems.

Abbreviation

Abbreviation: RTS, Rapid translation system; G-CSF, Granulocyte colony-stimulating factor; ORF, open reading frame; DMSO, dimethyl sulfoxide; MCS,multiple cloning site; RBS, ribosomal binding site; SAP, shrimp alkaline phosphatase; CECF, continuous exchange cell-free; HRP, horseradish peroxidise.