scholarly journals Cloning and expression of a chick liver glutathione S-transferase CL 3 subunit with the use of a baculovirus expression system

1992 ◽  
Vol 281 (2) ◽  
pp. 545-551 ◽  
Author(s):  
L H Chang ◽  
J Y Fan ◽  
L F Liu ◽  
S P Tsai ◽  
M F Tam
Keyword(s):  
Specific Activity ◽  
Sequence Similarity ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Relative Activity ◽  
Cloning And Expression ◽  
Glutathione S Transferase ◽  
Sds Page ◽  
Glutathione S Transferases ◽  
A Subunit

Glutathione S-transferase CL 3 subunits purified from 1-day-old-chick livers were digested with Achromobacter proteinase I and the resulting fragments were isolated for amino acid sequence analysis. An oligonucleotide probe was constructed accordingly for cDNA library screening. A cDNA clone of 1342 bases, pGCL301, encoding a protein of 26209 Da was isolated and sequenced. Including conservative substitutions, this protein has 75-79% sequence similarity to other Alpha family glutathione S-transferases. The coding sequence of pGCL301 was inserted into a baculovirus vector for infection of Spodoptera frugiperda (SF9) cells. The expressed protein has a high relative activity with ethacrynic acid (47% of the specific activity with 1-chloro-2,4-dinitrobenzene). The enzyme has a subunit molecular mass of 25.2 +/- 1.2 kDa (by SDS/PAGE), a pI of 9.45 and an absorption coefficient A1%1cm of 13.0 +/- 0.5 at 280 nm.

scholarly journals Glutathione S-transferases from the white-rot fungus, Phanerochaete chrysosporium

1997 ◽  
Vol 324 (1) ◽  
pp. 243-248 ◽  
Author(s):  
Caitriona A. DOWD ◽  
Catherine M. BUCKLEY ◽  
David SHEEHAN
Keyword(s):  
Molecular Mass ◽  
Phanerochaete Chrysosporium ◽  
Sequence Similarity ◽  
Gel Filtration ◽  
Preliminary Evidence ◽  
White Rot ◽  
White Rot Fungus ◽  
Glutathione S Transferase ◽  
Glutathione S Transferases ◽  
A Subunit

A glutathione S-transferase (GST) was purified to homogeneity from the white-rot fungus, Phanerochaete chrysosporium, by affinity chromatography on glutathione–agarose followed by Mono-Q ion-exchange FPLC. This protein immunoblotted with antisera to rat Theta class GST 5-5 and also showed N-terminal sequence similarity to the Theta class, including the presence of a conserved serine residue that has been specifically implicated in catalysis in this class [Wilce, Board, Feil and Parker (1995) EMBO J. 14, 2133–2143] and other residues conserved in plant sequences. Catalytic activity was found to be highly labile in the purified protein, although preliminary evidence for activity (approx. 120 m-units/mg) with 1,2-epoxy-3-(p-nitrophenoxy)propane was obtained in some preparations. The enzyme seems to be a dimer with a subunit molecular mass of 25 kDa by SDS/PAGE. The native molecular masses estimated by non-denaturing electrophoresis and by Superose-12 gel filtration were 58 and 45 kDa respectively. A second protein purified in this study also gave low level of activity with 1,2-epoxy-3-(p-nitrophenoxy)propane and had a subunit molecular mass of 28 kDa (native size 62–63 kDa), but did not immunoblot with any GST class and seemed to be N-terminally blocked.

scholarly journals Cloning and expression of haloacid dehalogenase gene from Bacillus cereus IndB1

2018 ◽  
Vol 22 (2) ◽  
pp. 55
Author(s):  
Enny Ratnaningsih ◽  
Idris Idris
Keyword(s):  
Recombinant Protein ◽  
Bacillus Cereus ◽  
Tertiary Structure ◽  
Specific Activity ◽  
Halogen Bond ◽  
Expression System ◽  
Industrial Sector ◽  
Cloning And Expression ◽  
Haloacid Dehalogenase ◽  
Organohalogen Compounds

Organohalogen compounds, widely used as pesticides in agriculture and solvents in the industrial sector, cause environmental pollution and health problems due to their toxicity and persistence. Numerous studies have been conducted on the biodegradation of organohalogen compounds, with many focusing on the use of dehalogenase from bacteria. Haloacid dehalogenase is a group of enzymes that cleaves the carbon-halogen bond in halogenated aliphatic acids. In a previous study, the bcfd1 gene encoded haloacid dehalogenase from Bacillus cereus IndB1 was successfully isolated and characterized. This research aimed to create an expression system of the bcfd1 gene by subcloning this gene into pET expression vector and to overexpress the gene in Escherichia coli BL21 (DE3). In addition, the recombinant protein was characterized to gain a better understanding of the catalytic action of this enzyme. A high expression of bcfd1 was obtained by inducing the culture at OD550 0.8–1.0  using 0.01 mM IPTG as determined by SDS-PAGE. Zymogram analysis proved that the recombinant protein possessed dehalogenase activity. Bcfd1 activity toward monochloroacetic acid (MCA) showed specific activity of 37 U/mg at 30°C, pH 9. The predicted tertiary structure of Bcfd1 was estimated has conserved α/ß hydrolase folding motif for haloacid dehalogenase superfamily.

scholarly journals Characterization of glutathione S-transferases in rat kidney. Alteration of composition by cis-platinum

1988 ◽  
Vol 252 (1) ◽  
pp. 127-136 ◽  
Author(s):  
G M Trakshel ◽  
M D Maines
Keyword(s):  
Specific Activity ◽  
Rat Kidney ◽  
Male Rats ◽  
Glutathione S Transferase ◽  
Aberrant Form ◽  
Glutathione S Transferases ◽  
Exchange Matrix ◽  
Rat Kidney Cytosol

We have developed chromatographic and mathematical protocols that allowed the high resolution of glutathione S-transferase (GST) subunits, and the identification of a previously unresolved GST monomer in rat kidney cytosol; the monomer was identified tentatively as subunit 6. Also, an aberrant form of GST 7-7 dimer appeared to be present in the kidney. This development was utilized to illustrate the response of rat kidney GST following cis-platinum treatment in vivo. Rat kidney cytosol was separated into three ‘affinity families’ of GST activity after elution from a GSH-agarose matrix. The affinity peaks were characterized by quantitative differences in their subunit and dimeric compositions as determined by subsequent chromatography on a cation-exchange matrix and specific activity towards substrates. By use of these criteria, the major GST dimers of affinity peaks were tentatively identified. The major GST dimers in peak I were GST 1-1 and 1-2, in affinity peak II it was GST 2-2, and in peak III they were GST 3-3 and 7-7. GST 3-6 and/or 4-6, which have not been previously resolved in kidney cytosol, were also present in peak II. Alterations in the kidney cytosolic GST composition of male rats were detected subsequent to the administration of cis-platinum (7.0 mg/kg subcutaneously, 6 days). This treatment caused a pronounced alteration in the GST profile, and the pattern of alteration was markedly different from that reported for other chemicals in the kidney or in the liver. In general, the cellular contents of the GSTs of the Alpha and the Mu classes decreased and increased respectively. It is postulated that the decrease in the Alpha class of GSTs by cis-platinum treatment may be related to renal cortical damage and the loss of GSTs in the urine. The increase in the Mu class of GSTs could potentially stem from a lowered serum concentration of testosterone; the latter is a known effect of cis-platinum treatment.

scholarly journals Partial characterization of natural and recombinant human soluble CD23

1992 ◽  
Vol 286 (3) ◽  
pp. 819-824 ◽  
Author(s):  
K Rose ◽  
G Turcatti ◽  
P Graber ◽  
S Pochon ◽  
P O Regamey ◽  
...  
Keyword(s):  
Insect Cells ◽  
Peptide Mapping ◽  
Sequence Similarity ◽  
Expression System ◽  
Protein A ◽  
Baculovirus Expression System ◽  
Partial Characterization ◽  
Disulphide Bonds ◽  
Baculovirus Expression

The purification to homogeneity of an active soluble 25 kDa fragment of CD23, produced in insect cells using the baculovirus expression system, is described. Peptide mapping and analysis by Edman degradation and mass spectrometry permitted partial characterization of the protein. A total of 165 out of 172 residues, including N-terminal and C-terminal regions, were mapped. The positions of the two disulphide bonds in the IgE-binding region were also determined: residue 110 is joined to residue 124, and residue 42 to residue 133. Natural CD23 25 kDa fragment was also analysed and found to possess the same disulphide bond arrangement. These results extend the previously noted sequence similarity with lectins to elements of secondary structure.

scholarly journals Expression and selective inhibition of the constitutive and inducible forms of human cyclo-oxygenase

1995 ◽  
Vol 305 (2) ◽  
pp. 479-484 ◽  
Author(s):  
J K Gierse ◽  
S D Hauser ◽  
D P Creely ◽  
C Koboldt ◽  
S H Rangwala ◽  
...  
Keyword(s):  
Competitive Inhibition ◽  
Specific Activity ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Selective Inhibition ◽  
Competitive Inhibitor ◽  
Chronic Inflammatory Disease ◽  
Dependent Inactivation ◽  
Inflammatory Drugs ◽  
Cox 1

The enzyme cyclo-oxygenase catalyses the oxygenation of arachidonic acid, leading to the formation of prostaglandins. Recently two forms of cyclo-oxygenase have been described: a constitutive (COX-1) enzyme present in most cells and tissues, and an inducible (COX-2) isoenzyme observed in many cells in response to pro-inflammatory cytokines. Constitutive and inducible forms of human cyclo-oxygenase (hCOX-1 and hCOX-2) were cloned and expressed in insect cells, utilizing a baculovirus expression system. hCOX-1 had a specific activity of 18.8 mumol of O2/mg with a Km of 13.8 microM for arachidonate and Vmax. of 1500 nmol of O2/nmol of enzyme, whereas hCOX-2 had a specific activity of 12.2 mumol of O2/mg with a Km of 8.7 microM for arachidonate and a Vmax. of 1090 nmol of O2/nmol of enzyme. Indomethacin inhibited both hCOX-1 and hCOX-2, whereas NS-398 and Dup-697 selectively inhibited hCOX-2. Both NS-398 and Dup-697 exhibited time-dependent inactivation of hCOX-2, as did indomethacin on both enzymes. The competitive inhibitor of hCOX-1, mefenamic acid, also displayed competitive inhibition of hCOX-2. These results demonstrate the ability to generate selective non-steroidal anti-inflammatory drugs (NSAIDs), which could provide useful improvement therapeutically in the treatment of chronic inflammatory disease.

Characterization of a nonphotosynthetic NADP-malic enzyme from germinating soybean cotyledons: interaction with a 94-kDa polypeptide identified as a lipoxygenase

2005 ◽  
Vol 83 (5) ◽  
pp. 529-538
Author(s):  
María Lorena Falcone Ferreyra ◽  
María Fabiana Drincovich ◽  
Carlos S Andreo ◽  
Florencio E Podestá
Keyword(s):  
Malic Enzyme ◽  
Specific Activity ◽  
Ionization Time ◽  
Protein Protein Interaction ◽  
Sds Page ◽  
Glycine Max L ◽  
A Subunit ◽  
High Enzymatic Activity ◽  
Germinated Soybean

The major NADP-malic enzyme (NADP-ME) isoform expressed in Glycine max L. cotyledons, leaves, and roots had a subunit molecular mass of 65 kDa and a pI of 6.9. The developmental profile of NADP-ME activity in cotyledons revealed a peak around day 7 post-imbibition (1.53 U·g–1 fresh mass, 33-fold that of day 1), declining markedly thereafter. NADP-ME was partially purified (33-fold) from 7-d germinated soybean cotyledons with a yield of 41% and a final specific activity of 2.8 U·mg–1. NADP-ME had an optimal pH of 7.3 and the Km values for NADP and L-malate were 33 µmol·L–1 and 0.78 mmol·L–1, respectively. The high enzymatic activity found in soybean cotyledons suggests that it plays an important role prior to the establishment of photosynthesis. NADP-ME co-purified with a 94-kDa polypeptide (p94) through various chromatographic steps. Non-denaturing electrophoresis followed by SDS-PAGE and immunoprecipitation assays indicated that NADP-ME is physically associated with p94. Matrix-assisted laser desorption and ionization – time of flight MS of p94 tryptic peptides identified p94 as a lipoxygenase. Although the association was verified by different approaches, the functional role for such interaction is still speculative. Possible hypothesis are discussed.Key words: germinating cotyledon, isozyme, lipoxygenase, NADP-malic enzyme, protein–protein interaction, soybean.

scholarly journals Human intestinal glutathione S-transferases

1989 ◽  
Vol 257 (2) ◽  
pp. 471-476 ◽  
Author(s):  
W H M Peters ◽  
H M J Roelofs ◽  
F M Nagengast ◽  
J H M van Tongeren
Keyword(s):  
Small Intestine ◽  
Large Intestine ◽  
Specific Activity ◽  
Distal Colon ◽  
Glutathione S Transferase ◽  
Distal Small Intestine ◽  
Cytosolic Glutathione ◽  
Glutathione S Transferases ◽  
Isoenzyme Composition ◽  
Specific Activities

Cytosolic glutathione S-transferases were purified from the epithelial cells of human small and large intestine. These preparations were characterized with regard to specific activities, subunit and isoenzyme composition. Isoenzyme composition and specific activity showed little variation from proximal to distal small intestine. Specific activities of hepatic and intestinal enzymes from the same patient were comparable. Hepatic enzymes were mainly composed of 25 kDa subunits. Transferases from small intestine contained 24 and 25 kDa subunits, in variable amounts. Colon enzymes were composed of 24 kDa subunits. In most preparations, however, minor amounts of 27 and 27.5 kDa subunits were detectable. Separation into isoforms by isoelectric focusing revealed striking differences: glutathione S-transferases from liver were mainly basic or neutral, enzymes from small intestine were basic, neutral and acidic, whereas large intestine contained acidic isoforms only. The intestinal acidic transferase most probably was identical with glutathione S-transferase Pi, isolated from human placenta. In the hepatic preparation, this isoform was hardly detectable. The specific activity of glutathione S-transferase showed a sharp fall from small to large intestine. In proximal and distal colon, activity seemed to be about equal. In the ascending colon there might be a relationship between specific activity of glutathione S-transferases and age of the patient, activity decreasing with increasing age.

scholarly journals Cloning and expression of maltooligosyltrehalose trehalohydrolase from Sulfolobus solfataricus DSM 1616 in Bacillus subtilis WB800

2020 ◽  
Vol 18 (2) ◽  
pp. 363-372
Author(s):  
Nguyen Tien Cuong ◽  
Nguyen Thi Hien Trang ◽  
Nguyen Thi Thao ◽  
Le Thanh Hoang ◽  
Nguyen Sy Le Thanh ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Recombinant Protein ◽  
Amylase Activity ◽  
Expression System ◽  
Sulfolobus Solfataricus ◽  
Cloning And Expression ◽  
Exchange Event ◽  
Sds Page ◽  
Dna Fragment ◽  
Conventional Expression

Maltooligosyltrehalose trehalohydrolase (MTHase) is an industrial enzyme for the production of trehalose. A DNA fragment of 1680 bp encoding for MTHase was cloned from Sulfobolus solfataricus DSM 1616 then fused with promoter acoA-amyE already amplified from pMSE3 vector by PCR to generate an expression cassette acoMTH. Afterward the cassette was inserted into pAC7 vector for expression of the gene in Bacillus subtilis WB800 – a conventional expression system. Gene MTH was inserted into the genome of B. subtilis WB800 by cross-exchange event of pAC7 vector with the host genome for expression of high quality and high quantity of extracellular recombinant protein. By crossing-exchange event at 3’amyE-5’amyE, the expressional cassette was integrated into B. subtilis WB800 genome. The expressional cassette was integrated into B. subtilis WB800 genome replacing 3’amyE-5’amyE, hindering the native amylase activity of the host. Expression of expected protein was confirmed by electrophoresis SDS-PAGE. From our results, it indicates that gene MTH was expressed successfully in B. subtilis WB800. After 0.5% acetoin induction for 48 h, the data showed that the protein with a molecular mass of ~64 kDa on SDS-PAGE was expressed. The level of recombinant protein in WBpAacoMTH was increased and reached 2.5%, 15.2% and 21.95%, respectively comparing with native B. subtilis WB800.

scholarly journals Cloning and expression of hepatitis E virus ORF2 as an immunogen protein in baculovirus expression system

2018 ◽  
Vol 5 (1) ◽  
pp. 23-26
Author(s):  
SA Sadeghi ◽  
M Shahanaghi ◽  
MR Aghasadeghi ◽  
F Motevalli ◽  
MR Amiran ◽  
...  
Keyword(s):  
Hepatitis E Virus ◽  
Expression System ◽  
Baculovirus Expression System ◽  
Hepatitis E ◽  
Cloning And Expression ◽  
Baculovirus Expression
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