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Journal : ANNALES BOGORIENSES

Overproduction and Purification of Soluble Recombinant Human Granulocyte Colony Stimulating Factor in Escherichia coli Using Thioredoxin as Fusion Agustiyanti, Dian Fitria; Retnoningrum, Debbie Sofie; Rachmawati, Heni; Fuad, Asrul Muhamad
ANNALES BOGORIENSES Vol 21, No 1 (2017): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.14203/ab.v21i1.294

Abstract

Recombinant human Granulocyte Colony Stimulating Factor (G-CSF) has been produced in a soluble form in Escherichia coli BL21 (DE3) as a fusion protein. The open reading frame of G-CSF was synthetically constructed in previous work and was codon optimized for best expression in E. coli. In this research, the gene was fused to thioredoxin (Trx) at the N-terminal in pET32 vector. The purpose of this research was to optimize the overproduction and purification processes to obtain high yield recombinant protein in soluble form, and to characterize the Trx-G-CSF fusion protein. Overproduction was performed using IPTG induction method for 3 and 6 hours. The protein was purified by Ni-NTA affinity chromatography and separated using gradient concentration of imidazole. The purified protein was then characterized by SDS-PAGE and Western Blot analysis. Further, enterokinase was used to separate G-CSF from the fusion protein. The purified form of G-CSF was subsequently characterized using Western Blot and mass spectrometry using MALDI-TOF. The results showed that the fusion protein was successfully produced in soluble part as much as 48.25% were obtained after 3 hours of induction. The yield of  fusion protein was 67.37%  from total protein (229.65  mg protein/L culture). The Western Blot analysis showed the G-CSF band at around 18.6 kDa. Mass spectrometry with MALDI-TOF/ TOF revealed that 25.86% of amino acid residue was recognized as part of human G-CSF sequence. 
Construction of an EPO (Human-Erythropoietin) Synthetic Gene Through a Recurvise-PCR Method Fuad, Asrul Muhamad; Gusdinar, Tutus; Retnoningrum, Debbie Sofie; Natalia, Dessy
ANNALES BOGORIENSES Vol 12, No 1 (2008): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (7152.292 KB) | DOI: 10.1234/60

Abstract

Human  erythropoietin (hEPO)  is an  important glycoprotein  in human  that is coded by a single gene named EPO  (eryhtopoietin). EPO  is  a  glycoprotein  hormone  that  promotes  erythropoiesis,  which  is  the  formation process of mature  red  blood  cell  (erythrocytes)  in  human  bodies.  It  is  widely used  for  treatment  of anemia  in patient  With  chronic  renal failure.  Therefore  EPO has  been classified  as hematopoietic cytokine. Recombinant hEPO  (rhEPO)  has  been  commercially  available,  such  a  Epogen.  It  is  produced  in  mammalian  cell, such  as CHO  (Chine  e hamster ovary) cells  for  the  reason of  its complex structure as a glyco-protein. In  an effort  to  use and optimize heterolgous EPO gene expression  in  an  alternative eukaryotic  host  cells  such  as  yeast, an  EPO­synthetic  gene  (EPOsyn)  was  constructed.  The  synthetic  gene  had  been designed to contain  optimaI Pichiapastoris codon usage . It had  been constructed by a  recursive-PCR method  in  two-step PCR reactions. The gene was assembled  from  8 single strands synthetic  oligonuclotides having an average  length of 90 nt with 20  to 30 overlap  region  between  two  adjacent  oligos. The  synthetic  gene  has  less  GC  content  (4-.3 1%)  compared  its native (human) gene (59.08%). The synthetic gene has  been cloned  in  pCR2.1  cloning plasmid and sequenced. From  8  independent clones,  it was revealed  that  the error  rate  was  1.59%,  in which  1.42% was due  to deletions and 0.17% due  to substitutions. Design of  the gene sequences, construction method and DNA sequence analysis of  the gene will  be discussed  in  this  paper.   Keywords: Human erythropoietin (hEPO), erythropoiesis  EPO­synthetic  gene, recursive-PCR, Pichiapastoris, hematopoietic cytokine.