Asrul Muhamad Fuad, Asrul Muhamad
Research Center for Biotechnology, Indonesian Institute of Sciences JL. Raya Jakarta - Bogor Km.46 Cibinong 16911 Bogor - Indonesia

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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.
Preparation of An scFv-Based Immunoliposome Specific towards Transferrin Receptor Kusharyoto, Wien; Handayani, Ira; Sari, Martha; Fuad, Asrul Muhamad
ANNALES BOGORIENSES Vol 18, No 2 (2014): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (664.3 KB) | DOI: 10.1234/99

Abstract

An ideal therapeutic for cancer would be one that selectively targets to tumor cells, is nontoxic to normal cells, and that could be systemically delivered, thereby reaching metastases as well as primary tumor. Immunoliposomes directed by monoclonal antibody or its fragments are promising vehicles for tumor targeted drug delivery. Transferrin receptors (TfR) levels are elevated in various types of cancer cells and considered to correlate with the aggressive or proliferative ability of tumor cells. Therefore, TfR levels can be elaborated as a prognostic tumor marker, and TfR is a potential target for drug delivery in the therapy of malignant cells. Here, we report the preparation of an anti-TfR single-chain antibody variable (scFv) immunoliposome for tumortargeted delivery vehicle. The cDNA encoding the variable heavy and light chain domains of the anti-TfRscFv antibody fragment was derived from the murine monoclonal antibody Clone E6, which is specific towards transferrin receptor. The gene encoding the anti-TfR scFv fragment was codon optimized for expression inEscherichia coli, subsequently synthesized, and cloned into the expression vector pJexpress404. The His6- tagged anti-TfR scFv fragment was expressed in E. coli and purified by means of immobilized metal-ion  affinity chromatography on TALON™ matrix. SDS-PAGE revealed that the scFv fragment had the size of approximately 27 kDa, which corresponded with the predicted size of the protein based on its amino acid sequence. Liposome containing 5% MPB-DOPE were prepared by ethanol injection method. Afterwards, the anti-TfR scFv fragments were covalently conjugated to the liposome to produce the anti-TfR scFv immunoliposome with the size of around 200 to 300 nm.
Construction and Expression of Immunotoxin Anti EGFRvIII scFv-HPR Conjugate in Pichia pastoris as A Targeted Drug Candidate for Cancer Therapy Yuliawati, Yuliawati; Soejoedono, Retno Damayanti; Fuad, Asrul Muhamad
ANNALES BOGORIENSES Vol 18, No 1 (2014): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | DOI: 10.1234/89

Abstract

Epidermal growth factor receptor variant III (EGFRvIII) is a mutant of EGFR lacking 267 amino acids (exon-2 through 7) within its extracellular domain, resulting in the formation of a new epitope as a tumorspecific target. EGFRvIII is commonly found in GBM, breast, ovarian, prostate, and lung carcinomas. Antibodies or part of antibodies (e.g. single chain variable fragment or scFv) with specific activity against this receptor have been developed. These antibodies are internalized into the cell after receptor binding. Ribonucleases can be cytotoxic due to their inherent ability to degrade RNA, therefore causing cell death via inhibition of protein synthesis. The aim of this research is to construct, clone and express an immunotoxin-based conjugate combining an anti-EGFRvIII scFv with a HPRmut (human pancreatic ribonuclease mutant variant) in Pichia pastoris. The anti-EGFRvIII scFv gene was cloned into yeast expression vector pPICZαA and fused with EGFP gene as a marker. The HPRmut gene was subsequently cloned at the C-terminal of the scFv::EGFP fusion, resulting in the scFv::EGFP::HPR fusion construct. The fusion construct was successfully obtained and nucleotide sequence of plasmid was verified. This construct was used to transform Pichia pastoris SMD 1168H. The gene fusion was successfully transformed and expressed in P. pastoris with a transformation efficiency of 102 cfu/μg DNA. The transformed yeasts were screened on agar media containing up to 1000 μg/ml zeocin. Yeast transformants showed green fluorescent due to the expression of EGFP gene. The recombinant proteins have been expressed and secreted from P. pastoris as showed by immunoblotting and SDS-PAGE analyses, then purified by affinity chromatography method. The selected yeast transformant produced at least 15.85 mg of purified protein per liter of yeast culture.
Comparison of Gene Expression Between Two Types of Anti-EGFRvIII ScFv Antibodies Having Different Variable Domain Orders in Escherichia coli Dewi, Kartika Sari; 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 | Full PDF (450.568 KB) | DOI: 10.14203/ab.v21i1.299

Abstract

Several studies reported that the expression of various kinds of Single-chain variable fragment (scFv) antibodies in Escherichia coli are significantly influenced by the order of their variable domains. To date, the effect of the order of variable domains in the expression of scFv antibodies against epidermal growth factor receptor variant III (EGFRvIII) has not been reported. This study aimed to compare the expression between VH-linker-VL and VL-linker-VH domain orders of the anti-EGFRvIII scFv antibodies in E. coli expression system. Recombinant plasmids inserted with DNA encoding scFv proteins were transformed into E. coli NiCo21(DE3) competent cells and characterized by colony PCR. The expression of scFv proteins was done by using optimum concentration of inducer. Total proteins, soluble periplasmic and cytoplasmic proteins, also extracellular proteins were isolated, subsequently characterized by SDS-PAGE, Slot Blot, and ImageJ software analyses. The antigen-binding activity of both scFvs proteins against EGFRvIII was observed. The results showed that the relative percentage of scFv expression with VH-linker-VL domain order is higher than that of VL-linker-VH in each compartment. Moreover, both of scFvs proteins have antigen-binding activity against EGFRvIII.
Constitutive Expression of Candida antarctica Lipase B (CALB) in Pichia pastoris Using pGAPZα Vector Wahyuni, Febriana Dwi; Fuad, Asrul Muhamad; Suharsono, Suharsono
ANNALES BOGORIENSES Vol 20, No 1 (2016): Annales Bogorienses
Publisher : Research Center for Biotechnology - Indonesian Institute of Sciences (LIPI)

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (461.586 KB) | DOI: 10.14203/ab.v20i1.225

Abstract

The CalBsyn gene was previously constructed synthetically to encode Candida antarctica lipase B (CALB). Lipase from CalBsyn gene is slightly different from that of wild type CALB (CALB-wt) where it has three amino acids substitutions at different positions, i.e. V210I, A281E, and V221D, to improve enzyme’s thermostability and catalytic efficiency. The CalBsyn gene was isolated from pJ912-CalBsyn vector by digestion using XhoI restriction enzyme. The CalBsyn gene then was ligated to pGAPZα expression vector and transformed into E. coli TOP10F’ in order to obtain recombinant vector pGAPZα-CalBsyn. The result showed that pGAPZα-CalBsyn recombinant vector was successfully transformed into E. coli TOP10F’ with transformation efficiency of 4.11 x 103 cfu/µg plasmid DNA. The pGAPZα-CalBsyn recombinant plasmid was successfully introduced into Pichia pastoris SMD1168H using electroporation method with transformation efficiency of 1.01 x 102 cfu/µg DNA. Qualitative lipase activity assays showed that transformed P. pastoris secreted recombinant lipase (CALB) and has lipolytic activity; while quantitative lipase activity assays showed that the lipase activity was 63.5 Units/ml in 48 hours. Analysis using SDS-PAGE showed that CALB protein was expressed successfully and the recombinant protein’s molecular size was approximately 45 kDa.
The Effect Of Temperature On Recombinant Human Granulocyte Colony Stimulating Factor Production By Pichia pastoris Expression System Adiredja, Yuliawati; Fuad, Asrul Muhamad
Indonesian Journal of Pharmacy Vol 29 No 2, 2018
Publisher : Faculty of Pharmacy Universitas Gadjah Mada, Yogyakarta, Skip Utara, 55281, Indonesia

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (1286.552 KB) | DOI: 10.14499/indonesianjpharm29iss2pp94

Abstract

Granulocyte colony stimulating factor (G-CSF) is a haematopoetic growth factor that functions as specific stimulator of the survival, proliferation, and differentiation of precursor cells of the neutrophilic granulocyte cell lineage as well as an activator of mature neutrophil function. The main objective of this work is to compare the effect of different temperature on the production of extracellular recombinant G-CSF in Pichia  pastoris. Cells were cultured for 72h in baffled shake-flasks at 20°C, 25°C, and 30°C in two different medium; buffered glycerol/methanol-complex medium (BMGY/BMMY) and buffered minimal glycerol/methanol (BMGH/BMMH) after methanol induction every 12h. Expressed recombinant hG-CSF in the methylotrophic yeast P. pastoris was analyzed with SDS-PAGE. The 23 kDa protein was secreted into the culture supernatant when induced with methanol. Production of recombinant G-CSF protein in P. pastoris at 30°C at 48h incubation after methanol induction every 12h is the highest in both complex and minimum medium.