scholarly journals SUBKLONING DAN ISOLASI GEN PENYANDI MIKRONEMA 3 (MIC-3) Toxoplasma gondii ISOLAT LOKAL

Molekul ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. 10
Author(s):  
Diana Indrasanti ◽  
Aris Haryanto ◽  
Wayan T. Artama
Keyword(s):  
Toxoplasma Gondii ◽  
Expression Vector ◽  
Recombinant Dna ◽  
Cell Infection ◽  
Recombinant Dna Technology ◽  
Specific Primers ◽  
Gene Encoding ◽  
E Coli ◽  
Local Isolate ◽  
Microneme Protein

Microneme protein (MIC) is one of proteins that belongs to excretory-secretory antigens (ESAs) of Toxoplasma gondii. Microneme 3 protein (MIC-3) is the protein that plays an important role in the invasion proccess during cell infection as a mediator attachment parasite to the host cell. The aim of this research is to clone mic3 (gene encoding for MIC-3) of T. gondii from local isolate using recombinant DNA technology by cloning mic3 in an expression vector. Deoxyribonucleic acid (DNA) from T. gondii tachyzoites was amplified by PuRe Taq RTG-PCR Beads using mic3 specific primers. Amplified DNA was double digested using EcoRV and HindIII restriction endonucleases and then purified using EZ-10 spin coloumn purification kit. The mic3 DNA was ligated into pET-32a(+) expression vector and transformated into Escherichia coli BL21. The results showed that recombinant mic3gene 4.2 kDa has been successfully performed by cloning gene encoding for MIC-3 protein of T. gondii local isolate into pET-32a(+) and transformed to E. coli BL21.

scholarly journals Cloning Gene Encoding Micronema 3 (Mic3) Protein of Tachyzoite Toxoplasma Gondii Local Isolate

2015 ◽  
Vol 10 (1) ◽  
Author(s):  
Wayan T. Artama ◽  
Ni Nyoman Ayu Dewi ◽  
Didik Tulus Subekti
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Dna ◽  
Computer Software ◽  
Gene Encoding ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Local Isolate ◽  
Dna Fragment ◽  
Polymerase Chain ◽  
Toxoplasma Gondii Infection

Microneme 3 (MIC3) protein tachyzoites Toxoplasma gondii is one of protein which plays an important roleduring cell host invasion. Gene encoding MIC3 protein has been studied and it was suggested a potent vaccinecandidate against Toxoplasma gondii infection. The aim of this research is to clone and sequence the gene encodingMIC3 protein of tachyzoites Toxoplasma gondii local isolate by amplification using polymerase chain reaction withspecific primers. The amplified DNA fragment was cloned into pGEM-T and transformed into E. coli XL-1 Blue byheat shock method. Recombinant plasmids were isolated using alkali lysis method and analyzed by digestionusing restriction endonuclease enzymes PstI, HindIII, NcoI and EcoRV. The recombinant plasmids then sequencedto find out the nucleotide sequence of insert gene by ABIPRISM 377 DNA Sequencer. The DNA sequence thenwere analyzed by computer software for alignment. The result showed that transformation in E. coli XL-1 Blue bypGEM-T produced one clone that was encoding MIC3 protein. Analysis of 489 bp from 5’ and 447 from 3’ of genesequence showed 97-98% homology with gene encoding for MIC3 protein of RH isolate.Keywords: MIC3 protein, Toxoplasma gondii, tachyzoite, recombinant DNA

scholarly journals Cloning and Sequencing cDNA Encoding for Rhoptry-2 Toxoplasma Gondii Tachyzoite Local Isolate

2015 ◽  
Vol 10 (2) ◽  
Author(s):  
Wayan T. Artama ◽  
Yulia Sari ◽  
Didik Tulus Subekti ◽  
Soenarwan Hery Poerwanto ◽  
Jarot Subandono
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Plasmid ◽  
Messenger Rna ◽  
Secretory Protein ◽  
Gene Encoding ◽  
Isolation System ◽  
Complementary Dna ◽  
E Coli ◽  
Local Isolate ◽  
Cell Target

Rhoptry protein belongs to an excretory and secretory antigens (ESAs) that play an important role during activepenetration of parasite into the cell target. This protein an able Toxoplasma gondii to actively penetrate targetedcell, meanwhile ESAs protein stimulates intracellular vacuole modification. It is, therefore, after the parasitesuccessfully enter the cell target then Granule (GRA) proteins are responsible for the formation of parasitophorusvacuole, which is protect the fusion with other intracellular compartments such as lysosomal vacuole. Consequently,this parasite is being able to survive and multiply at the cell target. The current study was aimed to clone andsequens cDNA encoding for ROP-2 of local isolated T. gondii tachizoite through DNA recombinant technique.Total ribonucleic acid (RNA) was isolated from tachyzoites of local isolated T. gondii that were grown up in Balb/c mice. Messenger RNA was isolated from total RNA using PolyAtract mRNA Isolation System. Messenger RNA wasused as a template for synthesis cDNA using Riboclone cDNA Synthesis System AMV-RT. EcoRI adaptor fromRiboclone EcoRI Adaptor Ligation System was added to Complementary DNA and than ligated to pUC19. Recombinantplasmid was transformed into E. coli (XL1-Blue). The transformed E. coli XL-1 Blue were plated on LB agarcontaining X-Gal, IPTG and ampicillin. Recombinant clones (white colony) were picked up and grown up in theLB medium at 37oC overnight. Expression of recombinant protein was analysed by immunoblotting in order toidentify cDNA recombinant wich is express ESA of T. gondii local isolate. Recombinant plasmid were isolatedusing alkalilysis method and were elektroforated in 1% agarose gel. The isolated DNA recombinant plasmid wascut using Eco RI and then sequenced through Big Dye Terminator Mix AB1 377A Sequencer using M13 Forward andM13 Reverse primers. The conclusion of this results showed that the recombinant clone was coding for excretoryand secretory protein which has molecular weight of 54 kDa. The DNA alignments of sequence from the clonedgene showed 97% homology with gene encoding for ROP-2 of T. gondii RH isolate.Keywords: Toxoplasma gondii, tachizoite, ESA, complementary DNA, ROP2

scholarly journals Heterologous Expression and Purification of ? Galactosidase Protein Using Affinity Chromatography

2013 ◽  
Vol 5 (3) ◽  
pp. 499-513
Author(s):  
M. Z. Alam ◽  
L. Ragionieri ◽  
M. A. S. Santos ◽  
A. Iqbal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heterologous Expression ◽  
Affinity Chromatography ◽  
Recombinant Dna ◽  
Expression System ◽  
Eukaryotic Expression ◽  
Lacz Gene ◽  
Recombinant Dna Technology ◽  
E Coli ◽  
Elution Buffer

Enzymes and other protein purification using recombinant DNA technology have become popular due to scarcity of natural protein. Saccharomyces cerevisiae is a demanding host, since it facilitates protein expression by its relative simplicity, safe organisms, inexpensive and has many properties of eukaryotic expression system. As an alternative host we express E. coli lacZ gene with GST tag in Saccharomyces cerevisiae and successfully purified from soluble extracts. The concentration of soluble GST-? galactosidase protein was approximately 0.57 mg/ml of elution buffer yielded from 50 ml yeast cell culture. The ?-galactosidase protein from insoluble extract was low due to the increasing solubility of GST tag. Keywords: ?-galactosidase; Heterologous expression; GST tag; Affinity chromatography. © 2013 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. doi: http://dx.doi.org/10.3329/jsr.v5i3.13820 J. Sci. Res. 5 (3), 499-513 (2013)  

scholarly journals VERIFIKASI cDNA T29 SEBAGAI KANDIDAT GEN PENGKODE PROTEIN Toxoplasma gondii DENGAN METODE SDS PAGE

2005 ◽  
Vol 11 (1) ◽  
pp. 61-66
Author(s):  
Ira Djajanegara ◽  
Wayan Artama ◽  
Retno Lestari ◽  
Sabar Pambudi
Keyword(s):  
Toxoplasma Gondii ◽  
Recombinant Plasmid ◽  
Restriction Site ◽  
Standard Procedure ◽  
Pcr Amplification ◽  
Gene Encoding ◽  
E Coli ◽  
Gene Coding ◽  
Sds Page ◽  
Encoding Protein

The process of cDNA construction from mRNA isolated from Toxoplasma gondii has been done. There were 7 candidates cDNA which one of them is called T29. Since Toxoplasma gondii is the cause of toxoplasmosis infection, cloning the gene encoding protein from this parasite provides an important tool for developing diagnostic kit for detection of toxoplasmosis. Digestion of the cDNA T29 with EcoRI which is the restriction site where the cDNA was inserted yielded a 1.862 bp fragment. The fragment was subcloned into E. coli expression vector pMal-p2x and transformed into E.coli strain TB1. Colonies of TB1 were grown on ampicillin plates and the recombinant plasmid was extracted using the standard procedure. The plasmid was digested using EcoRI and PstI, checked by PCR amplification using malE and M13/pUC primers. The recombinant plasmid was expressed in TB1 and the protein extracted was ran in SDS PAGE to observe the presence of the expressed protein. Based on the data from this experiment, there was no expression result of the expressed cDNA which was confirm by the PCR result. Therefore, it was concluded that cDNA T29 was not carrying the gene coding for protein from parasite Toxoplasma gondii.

scholarly journals Expression of gene encoding flavonol synthase isolating from trung du xanh tea (Camellia sinensis var. macrophylla) in E. coli

2020 ◽  
Vol 42 (1) ◽  
Author(s):  
Hoang Thi Thu Yen ◽  
Vu Thi Lan ◽  
Huynh Thi Thu Hue
Keyword(s):  
Camellia Sinensis ◽  
Expression Vector ◽  
Soluble Fraction ◽  
Soluble Form ◽  
Gene Encoding ◽  
Flavonol Synthase ◽  
E Coli ◽  
Insoluble Form

Common flavonols in plants including quercetin, kaempferol and myricetin are synthesized from dihydroflavonols (dihydroquercetin-DHQ, dihydrokaempferol-DHK and dihydromyricetin-DHM) by flavonol synthase (FLS). In tea, FLS has been shown to metabolize dihydroquercetin to quercetin. The FLS gene was cloned and sequenced from the cultivated tea (Camellia sinensis var. macrophylla) in Thai Nguyen province. In this study, we presented the results of optimizing and designing an expression vector for recombinant FLS (recombinant FLS-rFLS). The FLS gene was ligated completely to the pET32a (+) vector, then expressed in E. coli Rosetta1 and Rosetta2 strain. Using 1mM IPTG to induce the expression of rFLS at 37oC, rFLS was obtained with 52.83 kDa in size and existed predominantly as insoluble form. E. coli Rosetta1 pET32a (+)_FLSproduces rFLS in the soluble fraction than E. coli Rosetta2 pET32a (+)_FLS. Next, E. coli Rosetta1 pET32a (+)_FLSwas optimized for expression at temperatures of 30oC, 23oC and 16oC (24 and 48 hours). After being induced for expression with 1mM IPTG in 48 hours and cultured at 16oC, E. coli Rosetta1 strain containing pET32a (+) FLS produced the largest amount of rFLS in the soluble form. 

Synthesis of a Collagen Analog in Escherichia Coli Using Recombinant DNA Technology

1989 ◽  
Vol 174 ◽  
Author(s):  
Ina Goldberg ◽  
A. J. Salerno
Keyword(s):  
Escherichia Coli ◽  
Fusion Protein ◽  
Tandem Repeats ◽  
Recombinant Dna ◽  
Cellular Proteins ◽  
Recombinant Dna Technology ◽  
Synthetic Genes ◽  
E Coli ◽  
Bacterial Protease

AbstractA family of totally synthetic genes coding for multiple tandem repeats of the amino acid sequence (Gly-Pro-Pro) has been prepared and inserted into the Clal cloning site of the expression vector pJL6. A representative recombinant plasmid, pACI, with an insert of about 340 bp, was established in an Escherichia coli strain bearing a defective λ prophage, to study expression of the CII-collagen analog fusion protein produced from pACI upon heat induction. The in vivo levels of synthetic gene expression obtained showed that the fusion protein was synthesized in E. coli, but was labile compared to other cellular proteins. This degradation could be significantly reduced by the genetic inhibition of a bacterial protease system.

scholarly journals Cloning of cDNA Encoding GRA1 Protein of Tachyzoite Toxoplasma Gondii Local Isolate

2015 ◽  
Vol 10 (1) ◽  
Author(s):  
Erma Sulistyaningsih ◽  
Sukarti Moeljopawiro ◽  
Jarot Subandono ◽  
Wayan T. Artama
Keyword(s):  
Toxoplasma Gondii ◽  
Dna Sequences ◽  
Messenger Rna ◽  
Vaccine Candidate ◽  
Dna Encoding ◽  
Gene Encoding ◽  
Local Isolate ◽  
Ecori Restriction ◽  
Encoding Gene

Gene encoding GRA1 protein is potent DNA-vaccine candidate against toxoplasmosis. The aim of the researchwas to clone the gene encoding GRA1 protein of tachyzoite Toxoplasma gondii local isolate by DNA recombinanttechnology. Tachyzoite was grown in Balb/c mice in vivo. Messenger RNA was isolated from total RNA and itwas used to synthesis cDNA. Complementary DNA encoding GRA1 protein of tachyzoite Toxoplasma gondii localisolate was amplified and cloned in a prokaryote cloning vector. The recombinant GRA1-encoding gene was thendigesting using EcoRI restriction endonuclease and sequencing. The result showed that the recombinant GRA1-encoding gene consisted of DNA sequences encoding all signal peptide and mature peptide of GRA1 protein.Alignment of recombinant GRA1 sequence to gene encoding GRA1 protein of Toxoplasma gondii RH isolate showed100% homologous.Keywords: GRA1 protein, Toxoplasma gondii, tachyzoite, cloning, cDNA

scholarly journals Critical Factors Affecting the Success of Cloning, Expression, and Mass Production of Enzymes by Recombinant E. coli

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Md. Fakruddin ◽  
Reaz Mohammad Mazumdar ◽  
Khanjada Shahnewaj Bin Mannan ◽  
Abhijit Chowdhury ◽  
Md. Nur Hossain
Keyword(s):  
Mass Production ◽  
Recombinant Dna ◽  
Critical Factors ◽  
Molecular Tools ◽  
Recombinant Enzymes ◽  
Recombinant Dna Technology ◽  
Factors Affecting ◽  
E Coli ◽  
Industrial Level ◽  
Protein Enzyme

E. coli is the most frequently used host for production of enzymes and other proteins by recombinant DNA technology. E. coli is preferable for its relative simplicity, inexpensive and fast high-density cultivation, well-known genetics, and large number of compatible molecular tools available. Despite all these advantages, expression and production of recombinant enzymes are not always successful and often result in insoluble and nonfunctional proteins. There are many factors that affect the success of cloning, expression, and mass production of enzymes by recombinant E. coli. In this paper, these critical factors and approaches to overcome these obstacles are summarized focusing controlled expression of target protein/enzyme in an unmodified form at industrial level.

scholarly journals Proposition of a Novel Strategy for Creation of Entirely New Proteins

2021 ◽  
Author(s):  
Kenji Ikehara
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Recombinant Dna ◽  
Narrow Range ◽  
Amino Acid Sequences ◽  
Recombinant Dna Technology ◽  
Gene Encoding ◽  
Protein Functions ◽  
Novel Strategy ◽  
New Protein

Proteins having a variety of functions play many essential roles in maintaining various life activities in organisms. Various methods, by which new protein functions can be artificially produced, have progressed rapidly upon development in recombinant DNA technology and effective screening techniques. However, the obtainable scope of the new functions has been restricted in a narrow range, because only functions of presently existing proteins can be used. On the other hand, it has been considered that it would be impossible to create an entirely new protein, which does not show any meaningful homology with any other amino acid sequences of previously existing proteins. The reason is because one amino acid sequence for a protein cannot be selected out from an extraordinary large amino acid sequence diversity as ~10130. As a matter of course, it is impossible to design an amino acid sequence of a protein in advance and a gene encoding the protein cannot be also formed through random process. Nevertheless, extant organisms have generated a variety of entirely new proteins in some way to make full use of them. This means that extant organisms have equipped a mechanism with which entirely new proteins can be produced under the present core life system composed of protein, tRNA (genetic code) and gene. In this article, first I introduce the mechanism, with which entirely new proteins are created in extant organisms, and further propose a novel strategy for application of the mechanism to protein engineering through creation of entirely new proteins, which could contribute to development of various industries.

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