scholarly journals Optimizations of expression and purification of recombinant HIV-1 CRF01_AE p24 protein in Escherichia coli for development of immunodiagnostic assay

2015 ◽  
Vol 24 (1) ◽  
pp. 14-8
Author(s):  
Ade P.R. Simaremare ◽  
Budiman Bela ◽  
Andi Yasmon ◽  
Fera Ibrahim
Keyword(s):  
Escherichia Coli ◽  
Western Blot ◽  
Coli Strain ◽  
Expression And Purification ◽  
Purification System ◽  
P24 Protein ◽  
Native Condition ◽  
Different Strains ◽  
Hiv 1 ◽  
Induction Temperature

Background: Conventional method for confirmation of HIV infection is Western blot. However, it has limitations because of contamination by human cellular antigen and genetic diversity among the HIV-1 subtypes that show indeterminate result and inaccuracy for the diagnosis of different strains. Most of Western blot developed are based on HIV-1 B subtype. In Indonesia HIV-1 CRF01_AE subtype is dominantly circulated. Therefore, we optimized the expression, purification of the recombinant HIV-1 CRF01_AE p24 protein for development of immunodiagnostic assay.Methods: Optimization of protein expression in Escherichia coli strain BL21CP was performed including induction time, isopropyl-1-thio-d-galactopyranoside (IPTG) and immidazole consentrations, and induction temperature. Purification of the recombinant p24 protein was used by using Ni-NTA (Qiagen) purification system in native condition. Results: Expression and purification of HIV-1 CRF01_AE p24 protein have been performed. Confirmation of the recombinant protein by Western blot showed the expression and purification of recombinant p24 protein has been optimized well and reactive with sera of patients with HIV-1 CRF01_AE subtype positive.Conclusion: The recombinant HIV-1 CRF01_AE p24 protein has been expressed and purified successfully, and it is potential to be used as antigen for immunodiagnostic assay.

Large scale expression and purification of recombinant HIV-1 proteinase from Escherichia coli

1991 ◽  
Vol 21 (1-2) ◽  
pp. 127-136 ◽  
Author(s):  
Onkar M.P Singh ◽  
Dev S Baines ◽  
Richard M Hall ◽  
Norman M Gray ◽  
Malcolm P Weir
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Expression And Purification ◽  
Hiv 1

scholarly journals HIV-1 P24 EXPRESSION IN TRANSPLASTOMIC TOBACCO PLANTS

2020 ◽  
Vol 15 (2) ◽  
pp. 52-68
Author(s):  
Loc Tuong Phan ◽  
Ho Huu Nguyen ◽  
Thanh Thi Nguyen
Keyword(s):  
Western Blot ◽  
Southern Blot ◽  
Gel Filtration ◽  
Target Site ◽  
Gel Filtration Chromatography ◽  
Tobacco Plants ◽  
Transplastomic Tobacco ◽  
P24 Protein ◽  
High Concentrations ◽  
Hiv 1

Expression of HIV-1 p24 gene in chloroplasts was achieved in a tobacco variety V2 (Virginia TBE2). Through PCR and Southern blot analyses, it was demonstrated that the transgene integrated into the target site in the chloroplasts, between trnfM and trnG. Western blot results showed that HIV-1 p24 gene expressed in transplastomic tobacco plants. p24 protein accumulations were detected by ELISA in the range from 1.7% to 6.3% TSP and the high concentrations in the leaves near the top. p24 protein was purified by gel filtration chromatography demonstrated that the purification is 9.694 folds and the performance is 31.94%, however, protein p24 largely was inactive after purification.

120 IDENTIFICATION OF THE RELATIVELY PREDOMINANT BUFFALO INTERFERON tau ISOFORM AND ITS EXPRESSION IN ESCHERICHIA COLI

2014 ◽  
Vol 26 (1) ◽  
pp. 174 ◽  
Author(s):  
S. Saugandhika ◽  
H. N. Malik ◽  
D. K. Singhal ◽  
R. Singhal ◽  
A. Dubey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Western Blot ◽  
Signal Sequence ◽  
Rna Extraction ◽  
Coli Strain ◽  
Soluble Form ◽  
Interferon Tau ◽  
Amino Acid Homology ◽  
Ruminant Species

The maternal pregnancy recognition factor interferon tau (IFN-τ) is expressed in multiple isoforms in all pecoran ruminant species. Interferon-τ, as the first pregnancy signaling molecule, performs a significant role in implantation as well as establishment of pregnancy. Due to low reproductive efficiency of buffalo compared with bovine and IFN-τ being the key molecule of reproductive physiology in ruminants, the objective of our study was framed to identify the various IFN-τ transcripts in buffalo embryonic trophoblast cells, to know their relative abundance to identify the relatively predominant isoform, and lastly to clone and express it in a heterologous host. Following total cellular RNA extraction from primary trophectodermal cells, RT-PCR was performed using gene-specific primers designed against known bovine IFN-τ sequence. Cloning of the amplified product and screening of the recombinant colonies gave 13 distinct cDNA variants that encoded for 8 distinct buffalo IFN-τ isoforms. These buffalo IFN-τ isoforms have a greater nucleotide and amino acid homology with caprine IFN-τ (98–100% and 96–100%) than ovine (94–97% and 90–95%) and bovine (89.6–90.6% and 82–86%), respectively. The novel buffalo IFN-τ isoforms showed pronounced nucleotide and amino acid sequence identity with one another (99.1–99.8% and 98–99%) but only moderate identity with previously identified buffalo IFN-τ (90–92% and 82–86%). All the 13 transcript sequences were accepted in GenBank. Out of 8 isoforms, buffalo IFN-τ1 has been found to be the relatively predominant, which was subcloned into expression vector pET 22b without signal sequence from pJET cloning vector and expressed in competent BL21 (DE3) Escherichia coli strain. Expression of the recombinant protein in soluble form was induced by isopropyl β-D-1-thiogalactopyranoside (0.1 mM) at 30°C for 6 h. The recombinant BuIFN-τ obtained was confirmed by Western blot using anti-HIS antibody. A new 20-kDa protein was detected coinciding the molecular weight of IFN-τ reported earlier in literature. In conclusion, the current study revealed that there are 8 different isoforms of IFN-τ that are expressed in trophectodermal out-growths during early pregnancy of buffalo. Predominantly found isoform IFN-τ 1 was expressed in pET 22b vector, and recombinant soluble protein was confirmed by Western blot.

Expression and purification of soluble recombinant full length HIV-1 Pr55Gag protein in Escherichia coli

2014 ◽  
Vol 100 ◽  
pp. 10-18 ◽  
Author(s):  
William J. McKinstry ◽  
Marcel Hijnen ◽  
Hanumant S. Tanwar ◽  
Lindsay G. Sparrow ◽  
Sureshbabu Nagarajan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Full Length ◽  
Expression And Purification ◽  
Hiv 1

scholarly journals Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum

2007 ◽  
Vol 408 (2) ◽  
pp. 173-180 ◽  
Author(s):  
Paul W. Bowyer ◽  
Ruwani S. Gunaratne ◽  
Munira Grainger ◽  
Chrislaine Withers-Martinez ◽  
Sasala R. Wickramsinghe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmodium Falciparum ◽  
High Throughput ◽  
High Throughput Screening ◽  
Small Scale ◽  
Expression And Purification ◽  
Purification System ◽  
Pure Protein ◽  
Ic50 Values

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 μg·l−1 to >400 μg·l−1 by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale ‘piggyback’ inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values <50 μM, which demonstrate selectivity over human NMT1. Two of these compounds, when tested against cultured parasites in vitro, reduced parasitaemia by >80% at a concentration of 10 μM.

High-level expression and purification of mature HIV-1 protease in Escherichia coli under control of the araBAD promoter

1992 ◽  
Vol 37 (2) ◽  
Author(s):  
Alison Taylor ◽  
DavidP. Brown ◽  
Sunil Kadam ◽  
Mary Maus ◽  
WilliamE. Kohlbrenner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression And Purification ◽  
High Level Expression ◽  
High Level ◽  
Hiv 1

scholarly journals Temporal Appraisal and Molecular Characterization of Escherichia coli from Oyun River, Kwara State, Nigeria

2020 ◽  
pp. 56-63
Author(s):  
Olatunji Matthew Kolawole ◽  
Oluwasanmi Anuoluwapo Adeyemi
Keyword(s):  
Escherichia Coli ◽  
Molecular Characterization ◽  
Bacterial Load ◽  
Disease Outbreaks ◽  
Coli Strain ◽  
Microbial Count ◽  
Mean Values ◽  
E Coli ◽  
Risk Variants ◽  
Different Strains

Introduction: Escherichia coli, an indicator of feacal contamination has been proven to be the cause of several disease outbreaks in countries and continents around the world. Aim: To determine the genotypic variants of Escherichia coli present in Oyun River and provide information regarding the high-risk variants of E. coli in Oyun River. Study Design: The study cuts across the two seasons of Nigeria’s tropical climate weather, being the peak of the Harmattan season and the onset of the Rainy season. Three sampling sites (Jimba Oja; Unilorin Dam and Oyun in Ilorin, Kwara State) along the River course were examined for three months (February – April). Methodology: Heterotrophic counts, coli form counts and molecular characterization via PCR   using 16 sRNA primers, of water samples were done using standard microbiological and molecular methods. Results: Bacteriological results showed monthly mean values of microbial counts, ranged from 23.5 x 106 – 45.17 x 106 cfu/mL and total coli form count ranged from 53 cfu/100 mL to 256 cfu/100 mLs, both of which exceed the WHO standards of 100 cfu/mL for total microbial count and <1 cfu/100 mLs for total coliforms. A total of forty-eight coliforms were isolated, thirty two of which were Escherichia coli. Sequencing and BLAST analysis of eleven of the isolates using NCBI’s online database revealed five different strains. They include: Escherichia coli FAP1 genome (9.1%); Escherichia coli strain ST2747 (54.5%); Escherichia coli strain EADK4 (9.1%); Escherichia coli strain ST540 (18.2%) and Escherichia coli strain NCM3722 (9.1%). Correlation of results with previous studies showed that most of the strains identified were pathogenic. The E. coli strains isolated, coupled with the bacterial load, coliform count and some physicochemical parameters of Oyun River makes it unsafe for public consumption if not treated. Efforts therefore should be made to treat the water before use, while making frantic efforts to prevent further contamination of Oyun River.

scholarly journals MUTATIONS IN radA ARE RESPONSIBLE FOR ACQUIRED RADIO-RESISTANCE IN AN Escherichia coli STRAIN

2021 ◽  
Author(s):  
David Alcántara Díaz ◽  
Jorge Humberto Serment Guerrero ◽  
Gerardo Aguirre Escalona ◽  
Jorge Tonatiuh Ayala Sumuano
Keyword(s):  
Escherichia Coli ◽  
Gamma Radiation ◽  
Uv Light ◽  
Point Mutations ◽  
Coli Strain ◽  
Normal Strain ◽  
Wild Type ◽  
Different Strains ◽  
Strain Ab1157 ◽  
Selection Of

When bacteria are exposed to chronic or cyclic irradiation with ultraviolet (UV) light, it is observed that their resistance to this agent is increased by the selection of advantageous mutations under those conditions. UV light produces different damages in DNA, the repair of which is necessary to maintain the integrity of the genome. However, some damages can lead to such mutations when they are not properly repaired. In an earlier work, five subcultures of a wild-type Escherichia coli strain (PQ30) were cyclically irradiated with UV and different strains resistant to UV light and gamma radiation were obtained. In a preliminary mapping, different genes involved in their resistance to radiation were identified. In one of these strains, designated as IN801, the radA gene, the product of which is involved in recombinational DNA repair, was identified. In this work, cells from another wild-type strain (AB1157) were transformed with a plasmid (pUC19) that carries the radA gene from either PQ30 or IN801, in order to establish whether the radio-resistant phenotype can be transferred to a normal strain. Only cells that received the IN801 radA gene showed increased resistance to UV and gamma radiation. Further radA sequencing showed that the gene of IN801 acquired two-point mutations that replace two amino acids in the RadA protein, which most likely changed its enzymatic activities. These results confirm that radA participates in the radiation resistance of IN801.

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