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All Journal VALENSI Current Biochemistry INDONESIAN JOURNAL OF PHARMACY ANNALES BOGORIENSES Annales Bogorienses
Asrul Muhamad Fuad, Asrul Muhamad
Research Center for Biotechnology, Indonesian Institute of Sciences JL. Raya Jakarta - Bogor Km.46 Cibinong 16911 Bogor - Indonesia
Agriculture, Biological Sciences & Forestry Biochemistry, Genetics & Molecular Biology Chemical Engineering, Chemistry & Bioengineering Chemistry Education Immunology & microbiology Medicine & Pharmacology

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Factors Affecting Expression Level of Recombinant Human Epidermal Growth Factor in Escherichia coli BL21(DE3) and Size Exclusion Purification Thereof Pratiwi, Riyona Desvy; Agustina, Nabella; Aminah, Aminah; Fuad, Asrul Muhamad
Annales Bogorienses Vol. 25 No. 2 (2021): Annales Bogorienses
Publisher : BRIN

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Recombinant human epidermal growth factor (rhEGF) has been developed to provide the protein for therapeutic uses. Isopropyl β-d-1-thiogalactopyranoside (IPTG)-induced expression in Escherichia coli BL21(DE3) has shown the most effective system among other inducers systems in a similar host. However, suitable conditions related to IPTG concentration, incubation time, and incubation temperature are different depending on the amino acid content of the recombinant protein. This study aimed to evaluate the effects of various IPTG concentration, incubation time, and incubation temperature on rhEGF concentration. According to each analysis of those factors, induction with 0.05 mM IPTG for 2 h at 23°C was the most appropriate condition to obtain the highest concentration of rhEGF. The rhEGF was positively confirmed with a monoclonal antihEGF antibody and purified in high purity reaching 95.2%, yet recovery was low (1.44%) due to loss in fractions containing endogenous proteins. Therefore, further studies related to type of matrix, column length, and sample concentration in applying size exclusion chromatography are requested for higher recovery
Evaluation of Alternative Components in Growth Media of Lactobacillus brevis for Halal Probiotic Preparation Pratiwi, Riyona Desvy; Zanjabila, Sabighoh; Fairuza, Dian; Aminah, Aminah; Praharyawan, Swastika; Fuad, Asrul Muhamad
Annales Bogorienses Vol. 24 No. 1 (2020): Annales Bogorienses
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Probiotic has been widely used in functional food because of numerous advantages for health. MRS broth is commonly used as standard medium in studying lactobacilli. However, in some communities - like muslim and vegetarian society, components in MRS broth/medium become an issue. Beef extract and peptone – animal derived substances as nitrogen sources in the MRS medium should be avoided for the vegetarian. Meanwhile, for the muslim society, all components must be halal-certified including those animal derived ingredients. Therefore, several alternative sources for beef extract and peptone substitution were studied. Combination of alternative nitrogen sources was applied. In order to increase the effect of the alternative nitrogen sources, alternative carbon sources were also included. This is the first report about effects of L. brevis media components on cells growth to expression level of surface layer protein (Slp). Whey, lactose, sucrose, and galactose showed high contribution to L. brevis growth. However, the tested concentration of those substances were not sufficient to obtain equal bacterial growth and Slp expression than that of MRS broth. In addition, yeast extract appeared necessary to maintain cell wall and Slp expression.
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
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Human erythropoietin (hEPO) is an important glycoprotein in human that is coded by a single gene named EPO (erythropoietin). EPO is a glycoprotein hormone that promotes erythropoiesis, which is the formation process of mature red blood cell (erythr eyte ) 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 (Chinese hamster ovary) ceil for the reason of it· complex structure as a glyco-protein. In an effort to use and optimize heterologous EPO gene expression in an alternative eukaryotic host cells such as yeast, an EPO synthetic gene (EPOsyn) was constructed. The synthetic gene had been designed to contain optimal Pichia pastoris 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 oligonucleotides having an average length of 90 nt with 20 to 30 overlap region between two adjacent oligos. The synthetic gene has less GC content (45.31%) 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.
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
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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 tumor specific 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 P. 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. 
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
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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 tumor targeted 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 in Escherichia 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.
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
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The synthetic gene of CalBsyn was previously constructed to encode Candida antarctica lipase B (CALB). Lipase of CalBsyn gene is slightly different from wild type CALB (CALB-wt) where it has three amino acids substitutions at different positions, i.e. V210I, A281E, and V221D, in order to improve its thermostability and catalytic efficiency. The CalBsyn gene was isolated from pJ912-CalBsyn vector by digestion using XhoI restriction enzyme. The 1136 bp fragment of CalBsyn gene was then ligated to pGAPZα expression vector and transformed into Escherichia coli TOP10F’ to obtain recombinant vector pGAPZα-CalBsyn. The result show that pGAPZα-CalBsyn recombinant vector was successfully transformed into E. coli TOP10F’ with transformation efficiency of 4.11x103 cfu/μg plasmid DNA. The pGAPZα-CalBsyn recombinant plasmid was successfully introduced into Pichia pastoris SMD1168H using electroporation method with transformation efficiency of 1.01x102 cfu/μg DNA. Recombinant protein expression was analyzed in several selected P. pastoris recombinant strains. Qualitative lipase activity assays results show that transformed P. pastoris-produced extracellular recombinant lipase (CALB) showing lipolytic activity; while results of quantitative lipase activity assays show that this Pichia-derived lipase achieved an activity of 6.35 Units/mL within 48 hours. SDS-PAGE analysis confirms the succesfull expression of CALB protein with molecular size was approximately 45 kDa.
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
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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.
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
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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.
Co-Authors Adiredja, Yuliawati Agustina, Nabella Agustiyanti, Dian Fitria Agustiyanti, Dian Fitria Akbar, Nadhira Fathiaz Aminah Aminah Anak Agung Gede Sugianthara Aryani, Hanifah DEBBIE SOFIE RETNONINGRUM DESSY NATALIA Fairuza, Dian Febriana Dwi Wahyuni Handayani, Ira Handayani, Ira HENI RACHMAWATI Kartika Sari Dewi Kusharyoto, Wien Kusharyoto, Wien LAKSMI AMBARSARI Martha Sari Retno Damayanti Soejoedono Riyona Desvy Pratiwi, Riyona Desvy Suharsono Suharsono Swastika Praharyawan, Swastika TUTUS GUSDINAR Yuliawati Zanjabila, Sabighoh