OVERPRODUKSI, PURIFIKASI, DAN KARAKTERISASI HUMANGRANULOCYTE COLONY STIMULATING FACTOR (RHG-CSF) REKOMBINANPADA ESCHERICHIA COLI BL21(DE3)

• Main Author: Fitria Agustiyanti, Dian
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/79052
• Institution: Institut Teknologi Bandung

Granulocyte Colony Stimulating Factor (G-CSF) is a protein that stimulates the formation and maturation of neutrophil. Commercial product of G-CSF is presently produced in Escherichia coli in form of inclusion body. Previously, the open reading frame of G-CSF was codon optimized and chemically synthesized. The GCSF protein was produced as a fusion protein with thioredoxine (Trx) at its carboxyl terminus to obtain soluble G-CSF. The purpose of this research was to overproduce, purify, and characterize the Trx-G-CSF. To obtain soluble Trx-GCSF in high level, induction was done for 3 and 6 hours. Protein purification was performed by Nickle affinity column using imidazole with a gradual concentration in elution step. Crude extract and purified protein were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The purified protein was cleaved using Enterokinase to separate the Trx fragment from G-CSF part. The GCSF was characterized using Western blot and MALDO TOF/TOF MS analyses. The result showed that the highest yield of soluble protein, 288.5 mg/L culture was obtained at 3 h induction, Using 150 mM imidazole, the purified protein was obtained with the rendemen of 67.37 % (229.65 mg proteimL culture). Western blot method using specific antibody and MALDI-TOF/TOF MS analysis showed that the protein of 18.6 kDa was indeed G-CSF. The research reports the expression of soluble G-CSF in E. coli.