Purification by Affinity Chromatography of Glutathione Reductase (EC 1.6.4.2) from Escherichia coli and Characterization of such Enzyme

1984 ◽  
Vol 39 (9-10) ◽  
pp. 908-915 ◽  
Author(s):  
Anna M. Mata ◽  
M. Carmen ◽  
Juan López-Barea
Keyword(s):  
Escherichia Coli ◽  
Affinity Chromatography ◽  
Glutathione Reductase ◽  
Specific Activity ◽  
Total Yield ◽  
Coli Strain ◽  
Affinity Column ◽  
Heat Stable ◽  
Activity Loss ◽  
Fast Procedure

Abstract The glutathione reductase from Escherichia coli strain S33 was purified to homogeneity by a simple and fast procedure consisting of two affinity chromatography steps. After 40-80% ammonium sulfate fractionation, the enzyme was adsorbed to an N6-2′.5′-ADP-Sepharose affinity column from which it was specifically eluted by a 0 - 10 mᴍ NADP+ linear gradient. The enzyme was finally purified to homogeneity after a second affinity chromatography step in a C8-ATPR-Sepharose column, from which it was eluted by means of the same NADP+ gradient. Starting from 182 g of E. coli cells. 6.9 mg of pure enzyme was obtained after a 2632-fold purifi­cation, with a total yield of 63%. The pure enzyme showed a specific activity of 361 U/mg, and its absorption spectrum was characteristic of a flavoprotein. with an A272A450 of 7.84. The enzyme was a dimer with a molecular weight 109 000 and 40 Å hydrodynamic radius. The optimum pH were 7.5 and 4.5 with NADPH and NADH. respectively, as reductants. Apparent K′m values of 16, 377, and 66 μᴍ were determined at pH 7.5 for NADPH, NADH, and GSSG, respectively. Upon storage the enzyme was stable at pH values ranging from 7.5 to 9.5, being additionally stabilized by FAD. NADP+, dithiothreitol, or glycerol. The pure enzyme was quite heat stable, denaturing signifi­cantly only after 10 min at 70 0C. A marked activity loss was observed however, even at 0 °C, in the presence of 20 μᴍ NADPH. The enzyme was inactivated by low concentrations of para- hydroximercuribenzoate: the sensitivity towards such mercurial was greatly enhanced after reduction of the enzyme by NADPH.

scholarly journals Plasmid Coding for Drug Resistance and Production of Heat-Labile and Heat-Stable Toxins Harbored by an Escherichia coli Strain of Human Origin

1983 ◽  
Vol 39 (2) ◽  
pp. 970-973 ◽  
Author(s):  
M. Lourdes M. Silva ◽  
Isabel C. A. Scaletsky ◽  
M. Henriqueta L. Reis ◽  
M. Heloiza T. Affonso ◽  
Luiz R. Trabulsi
Keyword(s):  
Escherichia Coli ◽  
Drug Resistance ◽  
Coli Strain ◽  
Human Origin ◽  
Heat Stable ◽  
Heat Labile

scholarly journals Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 587-592 ◽  
Author(s):  
K M LeVan ◽  
E Goldberg
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Prokaryotic Expression ◽  
Specific Activity ◽  
Human Testis ◽  
Reading Frame ◽  
E Coli ◽  
Heat Stable ◽  
Prokaryotic Expression Vector ◽  
Tac Promoter

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

scholarly journals Prevalence of a Gene Encoding Adhesin Involved in Diffuse Adherence among Escherichia Coli Isolates in Pigs with Postweaning Diarrhea or Edema Disease

2003 ◽  
Vol 15 (4) ◽  
pp. 378-381 ◽  
Author(s):  
Seung-Kwon Ha ◽  
Changsun Choi ◽  
Chanhee Chae
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Coli Strain ◽  
Isolation Rate ◽  
Gene Encoding ◽  
Edema Disease ◽  
E Coli ◽  
Heat Stable ◽  
Fimbrial Adhesins ◽  
Postweaning Diarrhea

A total of 604 Escherichia coli strains isolated from weaned pigs with diarrhea or edema disease on 653 swine farms were screened for the presence of the adhesin involved in diffuse adherence (AIDA) gene by polymerase chain reaction (PCR). Escherichia coli isolates that carried AIDA genes were also tested by PCR for the detection of 5 fimbriae (F4, F5, F6, F18, and F41), 3 heat-stable (STa, STb, and EAST1) and 1 heat-labile (LT) enterotoxin, and Shiga toxin 2e (Stx2e) genes. Forty-five (7.5%) of the 604 E. coli isolates carried the gene for AIDA. Of these 45 isolates, 5 (11.1%) carried EAST1 genes only, 1 (2.2%) carried genes for at least one of the fimbrial adhesins, 12 (26.7%) carried genes for at least one of the toxins, and 27 (60%) carried genes for at least one of the fimbrial adhesins and toxins. Fifty-one percent of strains that carried AIDA genes carried Stx2e genes, and 40% of strains that carried AIDA genes carried F18ab. The isolation rate of enterotoxigenic E. coli strain carrying genes for AIDA was 87%, and the isolation rate of Shiga toxin-producing E. coli strain carrying genes for AIDA was 49%. AIDA may represent an important virulence determinant in pigs with postweaning diarrhea or edema disease.

scholarly journals Expression of a Temperature-Sensitive Esterase in a Novel Chaperone-Based Escherichia coli Strain

2004 ◽  
Vol 70 (8) ◽  
pp. 4499-4504 ◽  
Author(s):  
Manuel Ferrer ◽  
Tatyana N. Chernikova ◽  
Kenneth N. Timmis ◽  
Peter N. Golyshin
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Growth Temperature ◽  
Specific Activity ◽  
Maximum Growth ◽  
Maximum Growth Rate ◽  
Coli Strain ◽  
Temperature Sensitive ◽  
Psychrophilic Bacterium ◽  
E Coli

ABSTRACT A new principle for expression of heat-sensitive recombinant proteins in Escherichia coli at temperatures close to 4°C was experimentally evaluated. This principle was based on simultaneous expression of the target protein with chaperones (Cpn60 and Cpn10) from a psychrophilic bacterium, Oleispira antarctica RB8T, that allow E. coli to grow at high rates at 4°C (maximum growth rate, 0.28 h−1) (M. Ferrer, T. N. Chernikova, M. Yakimov, P. N. Golyshin, and K. N. Timmis, Nat. Biotechnol. 21:1266-1267, 2003). The expression of a temperature-sensitive esterase in this host at 4 to 10°C yielded enzyme specific activity that was 180-fold higher than the activity purified from the non-chaperonin-producing E. coli strain grown at 37°C (32,380 versus 190 μmol min−1 g−1). We present evidence that the increased specific activity was not due to the low growth temperature per se but was due to the fact that low temperature was beneficial to folding, with or without chaperones. This is the first report of successful use of a chaperone-based E. coli strain to express heat-labile recombinant proteins at temperatures below the theoretical minimum growth temperature of a common E. coli strain (7.5°C).

Construction of a Novel Recombinant Escherichia coli Strain Capable of Producing 1,3–propanediol

2011 ◽  
Vol 396-398 ◽  
pp. 2499-2502 ◽  
Author(s):  
Xiang Hui Qi ◽  
Qi Guo ◽  
Yu Tuo Wei ◽  
Hong Xu ◽  
Ri Bo Huang
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Gel Filtration ◽  
Coli Strain ◽  
Optimal Temperature ◽  
Recombinant Enzymes ◽  
Microbial Conversion ◽  
Gene Encoding ◽  
E Coli ◽  
Nickel Chelate

1, 3-propanediol (1, 3-PD) is biologically synthesized by glycerol dehydratase (GDHt) and 1, 3-propanediol dehydrogenase (PDOR). In present study, the gldABC gene, encoding GDHt from Klebsiella pneumoniae and the yqhD gene, encoding PDOR isoenzyme from E.coli BL21 were cloned and co-expressed in E.coli JM109 using plasmid pSE380. The over-expressed recombinant enzymes were purified by nickel-chelate chromatography combined with gel filtration to study the properties. Optimal temperature and pH of recombinant GDHt with specific activity of 85.8 U/mg were 45 °C and 9.0; and optimal temperature and pH of recombinant YqhD with specific activity of 80.0 U/mg were 37 °C, 7.0. The microbial conversion of 1,3-PD from glycerol by this recombinant E. coli strain was studied and the production of 1,3-PD was about 28.0 g/l.

scholarly journals Escherichia coli heat-stable enterotoxin: rapid method of purification and some characteristics of the toxin

1980 ◽  
Vol 28 (2) ◽  
pp. 469-474
Author(s):  
R Lallier ◽  
S Lariviere ◽  
S St-Pierre
Keyword(s):  
Escherichia Coli ◽  
Polyacrylamide Gel Electrophoresis ◽  
Coli Strain ◽  
Suckling Mice ◽  
Heat Stable ◽  
Yeast Extract Medium ◽  
Schiff Reagent ◽  
A Cell ◽  
Casamino Acids ◽  
Forced Aeration

In this study we used Escherichia coli strain F11(P155) of porcine origin. The heat-stable enterotoxin (ST) was produced with a batch fermentor under agitation (500 rpm) and forced aeration (5 liters/min) in Casamino Acids yeast extract medium containing 0.2% glucose. The pH varied from 7.2 to 7.8. The maximum amount of ST was obtained after 7 h of growth. ST was purified by ammonium sulfate precipitation, ultrafiltration on Amicon membranes, and chromatography in Bio-Gel P-4. The enterotoxin, which was purified approximately 1,000 times, was active at nanogram levels. On 20% polyacrylamide gel electrophoresis, ST exhibited an Rf of 0.6 ST was recovered from the gel slices by eluting in buffer and testing the activity in suckling mice, since no band appeared on the gel after staining with Coomasie brilliant blue R, Schiff reagent, or red oil. ST was resistant at pH 2 to 10 and at 100 degrees C for 15 min, but it was inactivated at 121 degrees C; it did not lose biological activity after treatment with pronase, lipase, or amylase. In suckling mice antiserum obtained from rabbits or goats immunized with ST neutralized the enterotoxin activity of a cell-free supernatant of purified ST.

scholarly journals Production and purification of heat-stable enterotoxin b from a porcine Escherichia coli strain.

1991 ◽  
Vol 59 (1) ◽  
pp. 198-203 ◽  
Author(s):  
J D Dubreuil ◽  
J M Fairbrother ◽  
R Lallier ◽  
S Larivière
Keyword(s):  
Escherichia Coli ◽  
Coli Strain ◽  
Heat Stable ◽  
Enterotoxin B

scholarly journals Recombinant FIX/X-Bp from the Japanese Habu Snake Is a Universal Ligand for Purification of Highly Carboxylated Factor IX Variants and Factor X

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1081-1081
Author(s):  
Oblaise Mercury ◽  
Lucy Liu ◽  
Ayman Ismail ◽  
Ming Zhang ◽  
Qi Lu ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Culture Medium ◽  
Specific Activity ◽  
Protein S ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Hek293 Cells ◽  
Affinity Column ◽  
Factor X

Abstract Background: The purification of vitamin K-dependent clotting factors typically involves multiple chromatographic steps, including an ion exchange-based pseudo-affinity step to enrich for species with sufficiently high gamma-carboxyglutamic acid (Gla) content to achieve maximal specific activity. Variants of these factors have been engineered to improve their pharmacokinetic properties by appending or inserting a variety of elements, including the Fc domain of IgG, unstructured hydrophilic peptides of defined amino acid composition (XTEN), albumin, and polyethylene glycol (PEG). In most cases, however, such modification alters both the hydrodynamic and electrostatic properties of the resulting molecule relative to those of the predicate molecule, thereby complicating their purification, particularly with regard to Gla enrichment by pseudo-affinity chromatography. Factor IX (FIX)- and factor X (FX)-binding protein (FIX/X-bp) isolated from the venom of the Japanese Habu snake (T. flavoviridis) has been shown to bind with high affinity and specificity to both FIX and FX, and structural studies have demonstrated that FIX/X-bp binds to the highly carboxylated calcium-bound forms of the Gla domains of these proteins. We therefore reasoned that FIX/X-bp could serve as a novel affinity ligand for rapid and simple purification of variants of FIX and FX with high specific activity. Aims: To generate and purify recombinant FIX/X-bp (rFIX/X-bp) and assess its utility for the purification of FIX, FIX-XTEN, FIX-albumin, and FX with high Gla content. Methods: A two-chained rFIX/X-bp molecule in which a polyhistidine tag was appended to one chain was generated by stable co-transfection of Chinese hamster ovary (CHO) cells. Culture medium was concentrated by tangential-flow filtration (TFF), and rFIX/X-bp was purified by one of two methods: 1) immobilized metal ion affinity chromatography (IMAC), followed by anion-exchange chromatography, or 2) affinity chromatography on immobilized FIX in calcium-containing buffer and subsequent elution in EDTA-containing buffer. The potent anticoagulant activity of rFIX/X-bp was verified by prothrombin time (PT) and activated partial thromboplastin time (APTT) assays, and its ability to bind to human FIX, FX, factor VII (FVII), protein S, and prothrombin was evaluated by biolayer interferometry. The affinity of rFIX/X-bp for FIX and FX was determined by surface plasmon resonance (SPR). An affinity column was then generated by chemical conjugation of rFIX/X-bp to NHS-activated Sepharose. Recombinant FIX, FIX-albumin, and FIX-XTEN were first affinity purified on IXSelect resin from the culture medium of transiently transfected HEK293 cells, and the resulting protein preparations, which were heterogeneous with regard to Gla content, were then applied to the rFIX/X-bp affinity column in calcium- or magnesium-containing buffer and eluted with EDTA-containing buffer. Activity was assessed by APTT assay, and Gla content was determined by mass spectrometric peptide mapping. Recombinant FX was purified from the culture medium of transiently transfected HEK293 cells by sequential barium citrate adsorption, anion exchange chromatography, and affinity chromatography on a rFIX/X-bp column. Results: In the presence of calcium or magnesium ions, rFIX/X-bp binds to FIX and FX with high affinity (KD≈ 10 pM), to a lesser extent to protein S and prothrombin, but not to FVII. FIX and FIX-albumin that had been affinity purified on a rFIX/X-bp column had specific activities that were consistent with published data and greater than 11 Gla residues per molecule. The Gla content of FX that had been affinity purified on a rFIX/X-bp column was 10 Gla residues per molecule (out of 11 possible). Conclusions: rFIX/X-bp is a universal ligand for the purification of highly carboxylated FX and FIX variants, including FIX-albumin and FIX-XTEN. Disclosures Mercury: Biogen: Employment. Liu:Biogen: Employment. Ismail:Biogen: Employment. Zhang:Biogen: Employment. Lu:Biogen: Employment. Cameron:Biogen: Employment. Goodman:Biogen: Employment. Culyba:Biogen: Employment. Ravindran Nair:Biogen: Employment. Holthaus:Biogen: Employment. Kulman:Biogen: Employment. Peters:Biogen: Employment.

Cloning and expression of the beta-D-phosphogalactoside galactohydrolase gene of Lactobacillus casei in Escherichia coli K-12

1982 ◽  
Vol 152 (3) ◽  
pp. 1138-1146
Author(s):  
L J Lee ◽  
J B Hansen ◽  
E K Jagusztyn-Krynicka ◽  
B M Chassy
Keyword(s):  
Escherichia Coli ◽  
Lactobacillus Casei ◽  
Specific Activity ◽  
Protein Product ◽  
Coli Strain ◽  
Cloning And Expression ◽  
E Coli ◽  
Lactose Metabolism ◽  
Gene Coding ◽  
K 12

Lactose metabolism in Lactobacillus casei 64H is associated with the presence of plasmid pLZ64. This plasmid determines both phosphoenolpyruvate-dependent phosphotransferase uptake of lactose and beta-D-phosphogalactoside galactohydrolase. A shotgun clone bank of chimeric plasmids containing restriction enzyme digest fragments of pLZ64 DNA was constructed in Escherichia coli K-12. One clone contained the gene coding for beta-D-phosphogalactoside galactohydrolase on a 7.9-kilobase PstI fragment cloned into the vector pBR322 in E. coli strain chi 1849. The beta-D-phosphogalactoside galactohydrolase enzyme isolated from E. coli showed no difference from that isolated from L. casei, and specific activity of beta-D-phosphogalactoside galactohydrolase was stimulated 1.8-fold in E. coli by growth in media containing beta-galactosides. A restriction map of the recombinant plasmid was compiled, and with that information, a series of subclones was constructed. From an analysis of the proteins produced by minicells prepared from transformant E. coli cells containing each of the recombinant subclone plasmids, it was found that the gene for the 56-kilodalton beta-D-phosphogalactoside galactohydrolase was transcribed from an L. casei-derived promoter. The gene for a second protein product (43 kilodaltons) was transcribed in the opposite direction, presumably under the control of a promoter in pBR322. The relationship of this second product to the lactose metabolism genes of L. casei is at present unknown.

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