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%.

scholarly journals Green Synthesis and Characterization of Silver Nanoparticles using Crude Extract of Crotalaria burhia

2021 ◽  
Vol 8 (4) ◽  
Author(s):  
Khalil Ahmad ◽  
Raeesa Noor ◽  
Muhammad Younus ◽  
Akram Chohan ◽  
Ume Habiba ◽  
...  
Keyword(s):  
Silver Nanoparticles ◽  
Antibacterial Activity ◽  
Zeta Potential ◽  
Green Synthesis ◽  
Crude Extract ◽  
Bacterial Infections ◽  
Synthesis And Characterization ◽  
Phytochemical Analysis ◽  
E Coli

Background: Appearance of antibiotic resistance has raised the demand to find alternative therapies and modified drug delivery system of medicinal plants to treat bacterial infections. Objective: The aim of this study is the green synthesis and characterization of silver nanoparticles by using crude extract of Crotalaria burhia and to evaluate their antibacterial potential. Methods: The roots and stems of plant were used to prepare the crude extract. The phytochemical analysis of different compounds in extract was performed. 1mM AgNO3 and different concentrations of plant extract were used for the green synthesis of silver nanoparticles. The particles size and zeta potential were measured by zeta sizer while surface morphology of silver nanoparticles was observed with Scanning Electron Microscope (SEM). The antibacterial activity of silver nanoparticles was performed by 96 well microdilution plate method. Results: The particle size and zeta potential of optimized formulation was 92 nm and -24.8 mV. The SEM analysis showed that silver nanoparticles are irregular and spherical shape. The antibacterial activity showed that MIC value of silver nanoparticles was lower for E. coli than S. aureus. Conclusion: Silver nanoparticles possess potent bactericidal activity against E. coli and moderate activity against S. aureus. It had been concluded that these nanoparticles can be used against multi-drug resistant bacterial infections.

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 Cloning and Expression of the Inositol Monophosphatase Gene from Methanococcus jannaschii and Characterization of the Enzyme

1998 ◽  
Vol 64 (7) ◽  
pp. 2609-2615 ◽  
Author(s):  
Liangjing Chen ◽  
Mary F. Roberts
Keyword(s):  
Gene Product ◽  
Heat Stability ◽  
Cloning And Expression ◽  
Hyperthermophilic Archaea ◽  
Methanococcus Jannaschii ◽  
Inositol Monophosphatase ◽  
E Coli ◽  
Low Concentrations ◽  
High Concentrations

ABSTRACT Inositol monophosphatase (EC 3.1.3.25 ) plays a pivotal role in the biosynthesis of di-myo-inositol-1,1′-phosphate, an osmolyte found in hyperthermophilic archaea. Given the sequence homology between the MJ109 gene product of Methanococcus jannaschii and human inositol monophosphatase, the MJ109 gene was cloned and expressed in Escherichia coli and examined for inositol monophosphatase activity. The purified MJ109 gene product showed inositol monophosphatase activity with kinetic parameters (Km = 0.091 ± 0.016 mM;V max = 9.3 ± 0.45 μmol of Pi min−1 mg of protein−1) comparable to those of mammalian and E. coli enzymes. Its substrate specificity, Mg2+ requirement, Li+inhibition, subunit association (dimerization), and heat stability were studied and compared to those of other inositol monophosphatases. The lack of inhibition by low concentrations of Li+ and high concentrations of Mg2+ and the high rates of hydrolysis of glucose-1-phosphate and p-nitrophenylphosphate are the most pronounced differences between the archaeal inositol monophosphatase and those from other sources. The possible causes of these kinetic differences are discussed, based on the active site sequence alignment between M. jannaschii and human inositol monophosphatase and the crystal structure of the mammalian enzyme.

CHARACTERIZATION OF PLUM POX VIRUS COAT PROTEIN EPITOPES USING FUSION PROTEINS EXPRESSED IN E. COLI

1998 ◽  
pp. 461-468 ◽  
Author(s):  
T. Candresse ◽  
F. Rafia ◽  
J. Dunez ◽  
M. Navratil ◽  
D. Boscia ◽  
...  
Keyword(s):  
Coat Protein ◽  
Fusion Proteins ◽  
Plum Pox Virus ◽  
E Coli ◽  
Virus Coat Protein ◽  
Virus Coat

scholarly journals Trigger Factor from the Psychrophilic Bacterium Psychrobacter frigidicola Is a Monomeric Chaperone

2008 ◽  
Vol 191 (4) ◽  
pp. 1162-1168 ◽  
Author(s):  
Sylvain Robin ◽  
Denisio M. Togashi ◽  
Alan G. Ryder ◽  
J. Gerard Wall
Keyword(s):  
Recombinant Protein ◽  
Quaternary Structure ◽  
Trigger Factor ◽  
Psychrophilic Bacterium ◽  
E Coli ◽  
Molar Ratios ◽  
A Cell ◽  
Protein Synthesis System ◽  
Cell Free Protein Synthesis

ABSTRACT In eubacteria, trigger factor (TF) is the first chaperone to interact with newly synthesized polypeptides and assist their folding as they emerge from the ribosome. We report the first characterization of a TF from a psychrophilic organism. TF from Psychrobacter frigidicola (TF Pf ) was cloned, produced in Escherichia coli, and purified. Strikingly, cross-linking and fluorescence anisotropy analyses revealed it to exist in solution as a monomer, unlike the well-characterized, dimeric E. coli TF (TF Ec ). Moreover, TF Pf did not exhibit the downturn in reactivation of unfolded GAPDH (glyceraldehyde-3-phosphate dehydrogenase) that is observed with its E. coli counterpart, even at high TF/GAPDH molar ratios and revealed dramatically reduced retardation of membrane translocation by a model recombinant protein compared to the E. coli chaperone. TF Pf was also significantly more effective than TF Ec at increasing the yield of soluble and functional recombinant protein in a cell-free protein synthesis system, indicating that it is not dependent on downstream systems for its chaperoning activity. We propose that TF Pf differs from TF Ec in its quaternary structure and chaperone activity, and we discuss the potential significance of these differences in its native environment.

Expression and characterization of a recombinant cysteine proteinase of Leishmania mexicana

2000 ◽  
Vol 347 (2) ◽  
pp. 383-388 ◽  
Author(s):  
Sanya J. SANDERSON ◽  
Kevin G. J. POLLOCK ◽  
James D. HILLEY ◽  
Morten MELDAL ◽  
Phaedria ST HILAIRE ◽  
...  
Keyword(s):  
Inclusion Bodies ◽  
Cysteine Proteinase ◽  
Native Enzyme ◽  
Leishmania Mexicana ◽  
E Coli ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Mature Enzyme ◽  
Pro Region

A major cysteine proteinase (CPB) of Leishmania mexicana, that is predominantly expressed in the form of the parasite that causes disease in mammals, has been overexpressed in Escherichia coli and purified from inclusion bodies to apparent homogeneity. The CPB enzyme, CPB2.8, was expressed as an inactive pro-form lacking the characteristic C-terminal extension (CPB2.8∆CTE). Pro-region processing was initiated during protein refolding and proceeded through several intermediate stages. Maximum enzyme activity accompanied removal of the entire pro-region. This was facilitated by acidification. Purified mature enzyme gave a single band on SDS/PAGE and gelatin SDS/PAGE gels, co-migrated with native enzyme in L. mexicana lysates, and had the same N-terminal sequence as the native enzyme. The procedure yielded > 3.5 mg of active enzyme per litre of E. coli culture.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

Antimicrobial Resistance and Virulence Characterization of E. Coli Isolated from Subclinical Mastitic Sheep and Goats

2018 ◽  
Vol 34 (3) ◽  
pp. 267-278
Author(s):  
Ashraf A. Abd El-Tawab ◽  
Mohamed G. Aggour ◽  
Fatma I. El- Hofy ◽  
Marwa M. Y. El- Mesalami
Keyword(s):  
Antimicrobial Resistance ◽  
Sheep And Goats ◽  
E Coli

Suitable Signal Peptides for Secretory Production of Recombinant Granulocyte Colony Stimulating Factor in Escherichia coli

2020 ◽  
Vol 14 (4) ◽  
pp. 269-282
Author(s):  
Sadra S. Tehrani ◽  
Golnaz Goodarzi ◽  
Mohsen Naghizadeh ◽  
Seyyed H. Khatami ◽  
Ahmad Movahedpour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Signal Peptide ◽  
Fusion Proteins ◽  
Inclusion Bodies ◽  
Clinical Aspects ◽  
Granulocyte Colony Stimulating Factor ◽  
Colony Stimulating Factor ◽  
E Coli ◽  
Secretory Production ◽  
Stimulating Factor

Background: Granulocyte colony-stimulating factor (G-CSF) expressed in engineered Escherichia coli (E. coli) as a recombinant protein is utilized as an adjunct to chemotherapy for improving neutropenia. Recombinant proteins overexpression may lead to the creation of inclusion bodies whose recovery is a tedious and costly process. To overcome the problem of inclusion bodies, secretory production might be used. To achieve a mature secretory protein product, suitable signal peptide (SP) selection is a vital step. Objective: In the present study, we aimed at in silico evaluation of proper SPs for secretory production of recombinant G-CSF in E. coli. Methods: Signal peptide website and UniProt were used to collect the SPs and G-CSF sequences. Then, SignalP were utilized in order to predict the SPs and location of their cleavage site. Physicochemical features and solubility were investigated by ProtParam and Protein-sol tools. Fusion proteins sub-cellular localization was predicted by ProtCompB. Results: LPP, ELBP, TSH, HST3, ELBH, AIDA and PET were excluded according to SignalP. The highest aliphatic index belonged to OMPC, TORT and THIB and PPA. Also, the highest GRAVY belonged to OMPC, ELAP, TORT, BLAT, THIB, and PSPE. Furthermore, G-CSF fused with all SPs were predicted as soluble fusion proteins except three SPs. Finally, we found OMPT, OMPF, PHOE, LAMB, SAT, and OMPP can translocate G-CSF into extracellular space. Conclusion: Six SPs were suitable for translocating G-CSF into the extracellular media. Although growing data indicate that the bioinformatics approaches can improve the precision and accuracy of studies, further experimental investigations and recent patents explaining several inventions associated to the clinical aspects of SPs for secretory production of recombinant GCSF in E. coli are required for final validation.

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