scholarly journals PRODUCTION AND CHARACTERIZATION OF RECOMBINANT PROTEINS CONTAINING BET V 2 ANTIGENIC EPITOPES OF BETULA PENDULA

2012 ◽  
Vol 9 (6) ◽  
pp. 28-31
Author(s):  
A V Chernysheva ◽  
V V Tyutyaeva ◽  
A V Pivovarova ◽  
I V Andreev ◽  
M N Sankov ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Inclusion Bodies ◽  
Birch Pollen ◽  
Elisa Test ◽  
Recombinant Allergen ◽  
Bacterial Cells ◽  
E Coli ◽  
Immunological Tests ◽  
Linear Epitopes

Aim of investigation. Production of immunologically active recombinant protein of Bet v 2 allergen ofbirch pollen. Materials and methods. mRNA was isolated from a sample ofbirch pollen. cDNA library was derived using SMART technology. Gene Bet v 2 was amplified by means of PCR with primers from the cDNA. The resulting PCR fragment of the gene was cloned into the vector pET29b(+). The recombinant protein Bet v 2 was expressed in cells E. coli, transformed with a plasmid. The recombinant protein was purified using NiNTA agarose. Immunological activity of the recombinant protein Bet v 2 was measured by ELISA and immunoblot methods. Results. The production system of the recombinant allergen Bet v 2 preparation suitable for immunological tests was developed during the research project. In the first phase allergen Bet v 2 gene was cloned from birch pollen collected in Russia. The gene was inserted into the vector pET29(+) for expression in bacterial cells. The expression cell strain E. coli was obtained with this plasmid. The synthesis of the recombinant protein that accumulates in inclusion bodies was activated in bacterial cells. The procedure of recombinant protein Bet v 2 isolation from inclusion bodies was developed by one round of chromatography purification. The recombinant protein isolation was carried out by chromatography on Niagarose. The highly purified preparation of the recombinant allergen was obtained as a result. The recognition of the recombinant protein Bet v 2 by sera varied in ELISA, indicating a different degree of patients sensitization to this allergen. In the immunoblot test the preparation was active only in 15% of cases. Apparently, reactive epitopes of the allergen are mainly conformational ones and are active in ELISA test, whereas linear epitopes, that are active in immunoblot, are in the minority. Conclusion.The system for production of recombinant allergen Bet v 2 preparation suitable for immunological tests has been developed.

scholarly journals Expression and Isolation of N-Terminal Truncated Human Recombinant Renalase in Prokaryotic Cells

2021 ◽  
Vol 4 (3) ◽  
pp. e00158
Author(s):  
V.I. Fedchenko ◽  
A.A. Kaloshin ◽  
S.A. Kaloshina ◽  
A.E. Medvedev
Keyword(s):  
Recombinant Protein ◽  
Signal Peptide ◽  
Extracellular Space ◽  
Inclusion Bodies ◽  
High Concentration ◽  
Genetic Construct ◽  
E Coli ◽  
Insoluble Form ◽  
Guanidine Chloride ◽  
Catalytic Functions

Renalase (RNLS) is a flavoproteinin which its N-terminal peptide (residues 1-17) has several important functions. In cells, it participates in the formation of the so-called Rossmanfold (residues 2-35), needed for «accommodation» of the FAD cofactor and for performing the catalytic functions of RNLS as a FAD-dependent oxidoreductase (EC 1.6.3.5). RNLS secretion into the extracellular space is accompanied by cleavage of this peptide. The resultant truncated extracellular RNLS cannot bind FAD and therefore performs various noncatalytic functions. In this work, we have performed expression the genetic construct encoding RNLS lacking its N-terminal signal peptide (tRNLS) in E. coli Rosetta (DE3) cells. The recombinant protein was accumulated in inclusion bodies in an insoluble form, which could be solubilized in the presence of a high concentration of urea or guanidine chloride. In contrast to full-length RNLS, which was effectively solubilized in the presence of 8 M urea, tRNLS was preferentially solubilized in the presence of 6 M guanidine chloride.

Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3)

2015 ◽  
Vol 110 ◽  
pp. 43-51 ◽  
Author(s):  
Sandeep Kumar ◽  
Kavish Kumar Jain ◽  
Anupam Singh ◽  
Amulya K. Panda ◽  
Ramesh Chander Kuhad
Keyword(s):  
Inclusion Bodies ◽  
Pectate Lyase ◽  
E Coli

Recombinant murine growth hormone from E. coli inclusion bodies: Expression, high-pressure solubilization and refolding, and characterization of activity and structure

2010 ◽  
Vol 26 (3) ◽  
pp. 743-749 ◽  
Author(s):  
Amber Haynes Fradkin ◽  
Carl S. Boand ◽  
Stephen P. Eisenberg ◽  
Mary S. Rosendahl ◽  
Theodore W. Randolph
Keyword(s):  
Growth Hormone ◽  
High Pressure ◽  
Inclusion Bodies ◽  
E Coli

Expression and Characterization of Recombinant Drosophila 6-pyruvoyl tetrahydropterin Synthase

Pteridines ◽  
1995 ◽  
Vol 6 (2) ◽  
pp. 58-62
Author(s):  
Young Shik Park ◽  
Nacksung Kim ◽  
Hajeong Kim ◽  
Dongkook Park ◽  
Jeongbin Yim
Keyword(s):  
Recombinant Protein ◽  
Isoelectric Point ◽  
Crude Extract ◽  
Fusion Proteins ◽  
Heat Stability ◽  
Native Enzyme ◽  
Metal Chelation ◽  
E Coli ◽  
Metal Chelating

Summary 6-Pyruvoyl tetrahydropterin synthase is involved in the synthesis of pteridine eye pigments in Drosophila. The purple gene which was known to be one of the target loci of the suppressor mutation su(sj2 has been identified to encode the enzyme, and its cDNA has been cloned recently. The cDNA encoding the 19.3 kDa subunit of the 6-pyruvoyl tetrahydropterin synthase was expressed as fusion proteins in E. coli. The recombinant protein was shown to be active and purified from the E. coli crude extract by metal-chelation chromatography. The fused metal-chelating oilgopeptide was removed by thrombin for further characterization. Apparent Km for the substrate dihydroneopterin triphosphate was determined to be 590 IlM, which was slightly higher than the value of the native enzyme. The isoelectric point of 6.4 was also different from the known value of 4.3 determined by the native enzyme. Heat stability and the stimulatory effect of reducing agents were similar to the native enzyme. The modification of cysteine residues in the recombinant enzyme, one of which is known to be conserved in human and rat enzymes, by iodoacetamide inhibited its activity by up to 80%.

Purification and Characterization of Recombinant sTRAIL Expressed in Escherichia coli

2004 ◽  
Vol 36 (2) ◽  
pp. 118-122 ◽  
Author(s):  
Xiao-Xia Xia ◽  
Ya-Ling Shen ◽  
Dong-Zhi Wei
Keyword(s):  
Inclusion Bodies ◽  
Tumor Cell Line ◽  
Single Step ◽  
Dilution Method ◽  
High Concentration ◽  
Purification And Characterization ◽  
E Coli ◽  
Metal Affinity ◽  
Soluble Trail

Abstract As a potential anti-tumor protein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has drawn considerable attention. This report presented the purification and characterization of soluble TRAIL, expressed as inclusion bodies in E. coli. sTRAIL inclusion bodies were solubilized and refolded at a high concentration up to 0.9 g/L by a simple dilution method. Refolded protein was purified to electrophoretic homogeneity by a single-step immobilized metal affinity chromatography. The purified sTRAIL had a strong cytotoxic activity against human pancreatic tumor cell line 1990, with ED50 about 1.5 mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had a structure similar to that of native protein with β-sheet secondary structure. This efficient procedure of sTRAIL renaturation may be useful for the mass production of this therapeutically important protein.

scholarly journals Pengaruh Jumlah Inokulum Sel Inang Bakteri E.coli BL21 (DE3) pLysS dan Waktu Overekspresi pada Produksi Protein Rekombinan Fim-C Salmonella typhi

2018 ◽  
Vol 4 (2) ◽  
pp. 98-106
Author(s):  
Muktiningsih - Nurjayadi ◽  
Izzatul Ilma Chairinnisa ◽  
Geta Putri Mentari ◽  
Dudi Hardianto ◽  
Asri Sulfianti ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Large Scale ◽  
Salmonella Typhi ◽  
Cell Number ◽  
Bacterial Cells ◽  
Typhoid Vaccine ◽  
Over Expression ◽  
Bacterial Cultures ◽  
Sds Page

Recombinant protein Fim-C S.typhi is a potential protein that can be used as an alternative typhoid vaccine and produced on a large scale. However, before entering into a large-scale stage, laboratory optimum data on the factors that affect the production process of Fim-C Salmonella typhi proteins are required. This study aims to obtain information regarding the effect of host cell number E.coli BL21 (DE3) pLysS and overexpression time on production of recombinant protein Fim-C Salmonella typhi as the basis for developing candidate of typhoid vaccine. The optimization process of overexpression was done by adding 0.5 mM IPTG (Isopropyl-β-D-thiogalactopyranoside) inducer into bacterial cultures of 2%, 4%, and 6% with 4, 5, and 6 hours over expression. The measurement of the concentration of Fim-C recombinant protein extracted samples were done by a spectrophotometric method used BCA Kit Assay Thermo ScientificTM with wavelength 590nm. The characterization of those protein extracts was performed using SDS-PAGE method. The results from the study concluded that the number of 4% E.coli bacterial cells and four-hour overexpression time are the optimum condition of Fim- C Salmonella typhi characterized by the presence of high-intensity bands at ± 31 kDa.  

scholarly journals Refolding and characterization of two G protein-coupled receptors purified from E. coli inclusion bodies

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247689
Author(s):  
Bastian Heim ◽  
René Handrick ◽  
Marcus D. Hartmann ◽  
Hans Kiefer
Keyword(s):  
G Protein ◽  
Inclusion Bodies ◽  
Protein Unfolding ◽  
G Protein Coupled Receptors ◽  
Orphan Receptor ◽  
Cubic Phase ◽  
E Coli ◽  
Sphingosine 1 Phosphate ◽  
G Protein Coupled

Aiming at streamlining GPCR production from E. coli inclusion bodies for structural analysis, we present a generic approach to assess and optimize refolding yield through thermostability analysis. Since commonly used hydrophobic dyes cannot be applied as probes for membrane protein unfolding, we adapted a technique based on reacting cysteins exposed upon thermal denaturation with fluorescent 7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). Successful expression, purification and refolding is shown for two G protein-coupled receptors (GPCR), the sphingosine-1-phosphate receptor S1P1, and the orphan receptor GPR3. Refolded receptors were subjected to lipidic cubic phase crystallization screening.

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