Heat stable proteinase fromPseudomonas fluorescensAH-70: purification by affinity chromatography on cyclopeptide antibiotics

1988 ◽  
Vol 55 (2) ◽  
pp. 217-226 ◽  
Author(s):  
Juan I. Azcona ◽  
Rosario Martín ◽  
Miguel A. Asensio ◽  
Pablo E. Hernández ◽  
Bernabé Sanz
Keyword(s):  
Affinity Chromatography ◽  
Specific Activity ◽  
Fold Increase ◽  
Total Activity ◽  
Purification Procedure ◽  
Extracellular Proteinase ◽  
Original Activity ◽  
Gramicidin S ◽  
Heat Stable ◽  
Sepharose 4B

SummaryA heat stable extracellular proteinase from the psychrotrophPseudomonas fluorescensAH-70 was purified to electrophoretic homogeneity by affinity chromatography on a gramicidin S–Sepharose-4B column. Bacitracin linked to Sepharose-4B was unable to retain any proteolytic activity, whereas the same antibiotic bound to AH-Sepharose-4B retained ~ 25% of the total activity. The purification procedure on the gramicidin S–Sepharose-4B column was easy to perform, fast and reproducible; it resulted in a 207-fold increase in the specific activity and a yield of 41% of the original activity. The purified enzyme was a monomer with a mol. wt of 33000. The enzyme hydrolysed whole casein and its fractions whereas no activity was observed against bovine serum albumin. The enzyme was a metalloproteinase. It was heat stable, havingD-values at 121, 135 and 150 °C of 3·8, 1·9 and 0·6 min respectively.

Purification and characterization of the extracellular proteinase ofPseudomonas fluorescensNCDO 2085

1986 ◽  
Vol 53 (3) ◽  
pp. 457-466 ◽  
Author(s):  
David J. Fairbairn ◽  
Barry A. Law
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Fold Increase ◽  
Exchange Chromatography ◽  
Purification And Characterization ◽  
Extracellular Proteinase ◽  
Original Activity ◽  
Heat Stable ◽  
D Values

SUMMAEYPseudomonas fluorescensNCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 °C and pH 7·0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3·5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5·40±0·05 and a mol. wt of 40200±2100. It is heat-stable having D-values at 74 and 140 °C of 1·6 and 1·0 min respectively; 40 and 70% of the original activity remained after HTST (74 °C/17 s) and ultra high temperature (140°C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from otherPseudomonasspp.

scholarly journals Thermostable glucose isomerase from psychrotolerant Streptomyces species

1970 ◽  
Vol 1 ◽  
pp. 6-10 ◽  
Author(s):  
Bidur Dhungel ◽  
Manoj Subedi ◽  
Kiran Babu Tiwari ◽  
Upendra Thapa Shrestha ◽  
Subarna Pokhrel ◽  
...  
Keyword(s):  
Specific Activity ◽  
Fold Increase ◽  
Glucose Isomerase ◽  
Enzyme Concentration ◽  
Maximal Velocity ◽  
Ph Optimum ◽  
Streptomyces Spp ◽  
Optimum Ph ◽  
Optimum Reaction ◽  
Sepharose 4B

Glucose isomerase (EC 5.3.1.5) was extracted from Streptomyces spp., isolated from Mt. Everest soil sample, and purified by ammonium sulfate fractionation and Sepharose-4B chromatography. A 7.1 fold increase in specific activity of the purified enzyme over crude was observed. Using glucose as substrate, the Michaelis constant (KM<) and maximal velocity (Vmax) were found to be 0.45M and 0.18U/mg. respectively. The optimum substrate (glucose) concentration, optimum enzyme concentration, optimum pH, optimum temperature, and optimum reaction time were 0.6M, 62.14μg/100μl, 6.9, 70ºC, and 30 minutes, respectively. Optimum concentrations of Mg2+ and Co2+ were 5mM and 0.5mM, respectively. The enzyme was thermostable with half-life 30 minutes at 100ºC.DOI: 10.3126/ijls.v1i0.2300 Int J Life Sci 1 : 6-10

scholarly journals A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

2020 ◽  
Author(s):  
Cecy Xi ◽  
Arianna Arianna Di Fazio ◽  
Naveed Nadvi ◽  
Karishma Patel ◽  
Michelle Xiang ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
High Purity ◽  
Specific Activity ◽  
Specific Binding ◽  
Exchange Chromatography ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
High Yield ◽  
Purification Procedure ◽  
Dipeptidyl Peptidase 4

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29-766) produced in insect cells. Purification used differential ammonium sulfate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor binding domain (RBD) were measured using surface plasmon resonance. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity &gt;30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

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 Hepatic microsomal Ca2+-dependent ATPase. Calmodulin-dependence and partial purification

1983 ◽  
Vol 214 (1) ◽  
pp. 69-75 ◽  
Author(s):  
P B Moore ◽  
N Kraus-Friedmann
Keyword(s):  
Affinity Chromatography ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Fold Increase ◽  
Partial Purification ◽  
Dependent Atpase

The hepatic microsomal fraction contains tightly bound calmodulin as demonstrated by affinity chromatography. When this calmodulin was partially removed by EGTA treatment (0.5 mM-EGTA), the uptake of 45Ca2+ by the microsomal vesicles was stimulated by added calmodulin and inhibited by trifluoperazine (TFP). The Ca2+-dependent ATPase was partially purified on a calmodulin column. This partial purification resulted in a 500-fold increase in the specific activity of the enzyme when measured in the presence of added calmodulin. Antibodies prepared against calmodulin prevented this stimulatory effect. The fraction eluted from the calmodulin column contained several protein bands indicating that the specific activity of the Ca2+-dependent ATPase is probably still underestimated. There are likely to be other calmodulin-sensitive processes present in the hepatic microsomal fraction.

scholarly journals A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Molecules ◽  
2020 ◽  
Vol 25 (22) ◽  
pp. 5392
Author(s):  
Cecy R Xi ◽  
Arianna Di Fazio ◽  
Naveed Ahmed Nadvi ◽  
Karishma Patel ◽  
Michelle Sui Wen Xiang ◽  
...  
Keyword(s):  
Surface Plasmon Resonance ◽  
Affinity Chromatography ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
High Purity ◽  
Specific Activity ◽  
Cell Surface Receptor ◽  
Spike Protein ◽  
High Yield ◽  
Purification Procedure

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29–766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.

Improved purification of human lactate dehydrogenase isoenzyme-3 and studies with its fluorescein isothiocyanate and biotin conjugates

1990 ◽  
Vol 36 (1) ◽  
pp. 59-64
Author(s):  
R N Weijers ◽  
R de Bruijn ◽  
J Mulder ◽  
H Kruijswijk
Keyword(s):  
Amino Acid ◽  
Lactate Dehydrogenase ◽  
Affinity Chromatography ◽  
B Lymphocytes ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Fluorescein Isothiocyanate ◽  
Molar Ratio ◽  
The Stability ◽  
Sepharose 4B

Abstract Lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) isoenzyme-3 (LD-3) has been isolated in milligram quantities from human erythrocytes. Using an improved procedure--which involves complete hemolysis of the erythrocytes, diethylaminoethyl (DEAE)-Sephacel column chromatography, and 5'-AMP-Sepharose 4B affinity chromatography--we obtained 23,000-fold purified isoenzyme from the crude hemolysate (overall yield about 90%). The final product was homogeneous on polyacrylamide disc gel electrophoresis and had a specific activity of about 435 kU/g. Its amino acid composition is presented. With the eventual aim to make visible and isolate IgA kappa antibody-secreting B lymphocytes, we developed reproducible methods for preparing fluorescein isothiocyanate isomer-1-conjugated LD-3 with a fluorescein/LD-3 molar ratio between 1.3 and 3.3, and biotinylated LD-3 with a biotin/LD-3 molar ratio between 1.3 and 2.5. In evaluating the stability of these two conjugates, we determined that they still can react with IgA kappa to form the IgA kappa (LD-3)2 complex.

scholarly journals Purification of calmodulin from Chlamydomonas: calmodulin occurs in cell bodies and flagella.

1980 ◽  
Vol 87 (3) ◽  
pp. 764-770 ◽  
Author(s):  
S E Gitelman ◽  
G B Witman
Keyword(s):  
Affinity Chromatography ◽  
Electrophoretic Mobility ◽  
Green Alga ◽  
Cyclic Nucleotide ◽  
Bovine Brain ◽  
Cyclic Nucleotide Phosphodiesterase ◽  
Polyacrylamide Gels ◽  
Relative Mobility ◽  
Heat Stable ◽  
Sepharose 4B

Calmodulin has been purified from cell bodies of the green alga Chlamydomonas by Ca++-dependent affinity chromatography on fluphenazine-Sepharose 4B. Calmodulin from this primitive organism closely resembles that from bovine brain in a number of properties, including (a) binding to fluphenazine in a Ca++-dependent, reversible manner, (b) functioning as a heat-stable, Ca++-dependent activator of cyclic nucleotide phosphodiesterase, and (c) electrophoretic mobility in SDS-polyacrylamide gels in both the presence and absence of Ca++, which causes a shift in the relative mobility of calmodulin. Calmodulin has also been identified by the criteria of phosphodiesterase activation and electrophoretic mobility in both the detergent soluble "membrane plus matrix" and the axoneme fractions of Chlamydomonas flagella. Calmodulin is not associated with the partially purified 12S or 18S dynein ATPases of Chlamydomonas. The presence of calmodulin in the flagellum suggests that it is involved in one or more of the Ca++-dependent activities of this organelle.

Isolation and purification of placental type alkaline phosphatase from a seminoma.

1987 ◽  
Vol 33 (2) ◽  
pp. 248-252
Author(s):  
G M Shameem ◽  
F Qadri
Keyword(s):  
Alkaline Phosphatase ◽  
Specific Activity ◽  
Cancer Tissue ◽  
Purification Procedure ◽  
Isolation And Purification ◽  
Alkaline Phosphatase Isoenzymes ◽  
Cancer Tissues ◽  
Sepharose 4B ◽  
Inhibition Studies ◽  
Blue Sepharose

Abstract An alkaline phosphatase (EC 3.1.3.1) of the placental type was isolated from a seminoma type of human testicular cancer tissue and was purified to homogeneity by sulfate-mediated chromatography on a column of Cibacron Blue Sepharose 4B. The purified enzyme had a specific activity of 40.6 kU per gram of protein and was obtained in a yield of 37%. The purification procedure used was simple and economical, and may be used to purify alkaline phosphatase isoenzymes from other cancer tissues. This is the first report of the purification of the enzyme in seminoma. Inhibition studies suggest that this enzyme is a Nagao variant rather than the Regan type reported in several cancer tissues.

scholarly journals Purification of alcohol dehydrogenase from Drosophila by general-ligand affinity chromatography

1979 ◽  
Vol 179 (3) ◽  
pp. 479-482 ◽  
Author(s):  
A J Brown ◽  
C Y Lee
Keyword(s):  
Drosophila Melanogaster ◽  
Alcohol Dehydrogenase ◽  
Affinity Chromatography ◽  
Good Yield ◽  
Purification Procedure ◽  
Ligand Affinity ◽  
Novel Technique ◽  
Affinity Ligand ◽  
Sepharose 4B ◽  
Homogeneous Preparation

A method for the purification of alcohol dehydrogenase from Drosophila melanogaster is described. The method makes use of 8-(6-aminohexyl)amino-5′-AMP, immobilized on Sepharose 4B, as an affinity ligand. Since alcohol dehydrogenase from Drosophila shows weak affinity for this column, a novel technique was developed to separate alcohol dehydrogenase from both unbound proteins and more strongly bound enzymes. The purification procedure is simple to operate and give a homogeneous preparation in good yield after only three steps.

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