scholarly journals Expression, purification and kinetic properties of human recombinant phospholipase C delta 3.

1997 ◽  
Vol 44 (2) ◽  
pp. 221-229 ◽  
Author(s):  
T Pawełczyk ◽  
A Matecki
Keyword(s):  
Phospholipase C ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Cation Exchange Chromatography ◽  
E Coli ◽  
His Tag ◽  
Absolute Requirement ◽  
Maximal Activation ◽  
Regulatory Properties

To obtain sufficient quantities of pure phospholipase C delta 3 (PLC delta 3) necessary for structural and kinetic studies, cDNA of human fibroblast PLC delta 3 was cloned in the pPROEX-1 vector, expressed in E. coli cells as a (6 x His) fusion protein and purified to homogeneity. From 1 L of E. coli culture 8 mg of pure PLC delta 3 was obtained by a two step purification procedure, which includes phosphocellulose and Mono S cation exchange chromatography. The presence of His tag did not affect the catalytic and regulatory properties of PLC delta 3. The K(app) for PIP2 was 142 +/- 11 and 156 +/- 12 microM for His.PLC delta 3 and PLC delta 3, respectively. Recombinant PLC delta 3 showed an absolute requirement for Ca2+. Increasing the free Ca2+ concentration from 0.2 to 0.5 microM resulted in a sharp increase in enzyme activity. In comparison with human recombinant PLC delta 1 the delta 3 isoenzyme was more sensitive to low Ca2+ concentration. The Ca2+ concentration yielding maximal activation of PLC delta 1 and PLC delta 3 was 10 and 1 microM, respectively. The activity of PLC delta 3 was stimulated by polyamines and by basic proteins such as protamine, histone and mellitin. PLC delta 3 was activated most effectively by spermine and histone but the extent of this activation was lower than for PLC delta 1. The data presented indicate that the expression of PLC delta 3 in E. coli cells permits to obtain active enzyme. The catalytic and regulatory properties of PLC delta 3 are similar to those of PLC delta 1.

scholarly journals Structural and Kinetic Properties of Nonglycosylated Recombinant Penicillium amagasakienseGlucose Oxidase Expressed in Escherichia coli

1998 ◽  
Vol 64 (4) ◽  
pp. 1405-1411 ◽  
Author(s):  
Susanne Witt ◽  
Mahavir Singh ◽  
Henryk M. Kalisz
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Pcr Amplification ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Kinetic Properties ◽  
Peptide Fragments ◽  
Thermal Stabilities ◽  
E Coli

ABSTRACT The gene coding for Penicillium amagasakiense glucose oxidase (GOX; β-d-glucose; oxygen 1-oxidoreductase [EC1.1.3.4 ]) has been cloned by PCR amplification with genomic DNA as template with oligonucleotide probes derived from amino acid sequences of N- and C-terminal peptide fragments of the enzyme. RecombinantEscherichia coli expression plasmids have been constructed from the heat-induced pCYTEXP1 expression vector containing the mature GOX coding sequence. When transformed into E. coli TG2, the plasmid directed the synthesis of 0.25 mg of protein in insoluble inclusion bodies per ml of E. coli culture containing more than 60% inactive GOX. Enzyme activity was reconstituted by treatment with 8 M urea and 30 mM dithiothreitol and subsequent 100-fold dilution to a final protein concentration of 0.05 to 0.1 mg ml−1 in a buffer containing reduced glutathione-oxidized glutathione, flavin adenine dinucleotide, and glycerol. Reactivation followed first-order kinetics and was optimal at 10°C. The reactivated recombinant GOX was purified to homogeneity by mild acidification and anion-exchange chromatography. Up to 12 mg of active GOX could be purified from a 1-liter E. coli culture. Circular dichroism demonstrated similar conformations for recombinant and native P. amagasakiense GOXs. The purified enzyme has a specific activity of 968 U mg−1 and exhibits kinetics of glucose oxidation similar to those of, but lower pH and thermal stabilities than, native GOX from P. amagasakiense. In contrast to the native enzyme, recombinant GOX is nonglycosylated and contains a single isoform of pI 4.5. This is the first reported expression of a fully active, nonglycosylated form of a eukaryotic, glycosylated GOX inE. coli.

Purification, Kinetic Properties, and Intracellular Concentration of SpoIIE, an Integral Membrane Protein That Regulates Sporulation in Bacillus subtilis

1999 ◽  
Vol 181 (10) ◽  
pp. 3242-3245 ◽  
Author(s):  
Isabelle Lucet ◽  
Rainer Borriss ◽  
Michael D. Yudkin
Keyword(s):  
Bacillus Subtilis ◽  
Kinetic Studies ◽  
Integral Membrane Protein ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Kinetic Properties ◽  
Bifunctional Protein ◽  
Molar Basis ◽  
Quantitative Immunoblotting

ABSTRACT SpoIIE is a bifunctional protein which controls ςFactivation and formation of the asymmetric septum in sporulatingBacillus subtilis. The spoIIE gene of B. subtilis has now been overexpressed in Escherichia coli, and SpoIIE has been purified by anion-exchange chromatography and affinity chromatography. Kinetic studies showed that the rate of dephosphorylation of SpoIIAA-P by purified SpoIIE in vitro was 100 times greater, on a molar basis, than the rate of phosphorylation of SpoIIAA by SpoIIAB. The intracellular concentrations of SpoIIE and SpoIIAB were measured by quantitative immunoblotting between 0 and 4 h after the beginning of sporulation. The facts that these concentrations were very similar at hour 2 and that SpoIIE could be readily detected before asymmetric septation suggest that SpoIIE activity may be strongly regulated.

scholarly journals Adenylate metabolism in the heart. Regulatory properties of rabbit cardiac adenylate deaminase

1979 ◽  
Vol 182 (2) ◽  
pp. 361-366 ◽  
Author(s):  
R Barsacchi ◽  
M Ranieri-Raggi ◽  
C Bergamini ◽  
A Raggi
Keyword(s):  
Skeletal Muscle ◽  
Kinetic Properties ◽  
Amp Deaminase ◽  
Alkaline Ph ◽  
Muscle Enzyme ◽  
Adenylate Deaminase ◽  
Activated State ◽  
Maximal Activation ◽  
Regulatory Properties

The kinetic properties of a 300-fold purified cardiac AMP deaminase were studied and compared with those of the corresponding enzyme from skeletal muscle. The heart enzyme is activated by ATP and less efficiently by ADP, and is inhibited by Pi, phosphocreatine and GTP. ATP, even at micromolar concentrations, is able to abolish the effects of the inhibitors. The affinity of the enzyme for AMP is low in the absence of activators (Km 3.1 mM), but, in the presence of ATP, becomes as high as that of skeletal-muscle AMP deaminase (Km 0.4 mM). The maximal activation by ATP is observed at alkaline pH (pH 7.5-8.0). Under the same conditions ATP is maximally inhibitory for skeletal-muscle enzyme. These results suggest that AMP deaminase in the heart is always in the activated state, whereas in skeletal muscle the enzyme is active only during exhaustive contractions.

scholarly journals Biochemical Characterization of a β-Galactosidase with a Low Temperature Optimum Obtained from an Antarctic Arthrobacter Isolate

2003 ◽  
Vol 185 (18) ◽  
pp. 5473-5482 ◽  
Author(s):  
James A. Coker ◽  
Peter P. Sheridan ◽  
Jennifer Loveland-Curtze ◽  
Kevin R. Gutshall ◽  
Ann J. Auman ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Thermal Adaptation ◽  
Kinetic Studies ◽  
E Coli ◽  
His Tag ◽  
Dry Valley ◽  
Diamino Acid ◽  
Positive Isolate ◽  
Rapid Purification

ABSTRACT A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 β-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18°C and 50% activity remaining at 0°C) and heat labile (inactivated within 10 min at 37°C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both β-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-β-d-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20°C than the comparable LacZ β-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4°C but dissociated into inactive monomers at 25°C. Comparisons of family 2 β-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.

scholarly journals Generation of Mature Nα-Terminal Acetylated Thymosinα1 by Cleavage of Recombinant Prothymosinα

2013 ◽  
Vol 2013 ◽  
pp. 1-8 ◽  
Author(s):  
Bo Liu ◽  
Xin Gong ◽  
Shaohong Chang ◽  
Peng Sun ◽  
Jun Wu
Keyword(s):  
Mass Spectrometry ◽  
High Performance Liquid Chromatography ◽  
High Performance ◽  
Expression System ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Reverse Phase ◽  
E Coli ◽  
Amino Acid Peptide

Nα-terminal acetylation of peptides plays an important biological role but is rarely observed in prokaryotes. Nα-terminal acetylated thymosinα1 (Tα1), a 28-amino-acid peptide, is an immune modifier that has been used in the clinic to treat hepatitis B and C virus (HBV/HCV) infections. We previously documented Nα-terminal acetylation of recombinant prothymosinα(ProTα) inE. coli. Here we present a method for production of Nα-acetylated Tα1 from recombinant ProTα. The recombinant ProTαwas cleaved by human legumain expressed inPichia pastoristo release Tα1in vitro. The Nα-acetylated Tα1 peptide was subsequently purified by reverse phase and cation exchange chromatography. Mass spectrometry indicated that the molecular mass of recombinant Nα-acetylated Tα1 was 3108.79 in, which is identical to the mass of Nα-acetylated Tα1 produced by total chemical synthesis. This mass corresponded to the nonacetylated Tα1 mass with a 42 Da increment. The retention time of recombinant Nα-acetylated Tα1 and chemosynthetic Nα-acetylated Tα1 were both 15.4 min in RP-high performance liquid chromatography (HPLC). These data support the use of anE. coliexpression system for the production of recombinant human Nα-acetylated Tα1 and also will provide the basis for the preparation of recombinant acetylated peptides inE. coli.

scholarly journals Chemical identity of tryptensin with angiotensin

1980 ◽  
Vol 187 (3) ◽  
pp. 647-653 ◽  
Author(s):  
K Arakawa ◽  
M Yuki ◽  
M Ikeda
Keyword(s):  
Amino Acid ◽  
Thin Layer ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Human Plasma Protein ◽  
Plasma Protein Fraction ◽  
Layer Chromatography

Tryptensin, a vasopressor substance generated from human plasma protein fraction IV-4 by trypsin, has been isolated and the amino acid composition analysed. The procedures used for the isolation were: (a) adsorption of the formed tryptensin on Dowex 50W (X2; NH4+ form); (b) gel filtration through Sephadex G-25; (c) cation-exchange chromatography on CM-cellulose; (d) anion-exchange chromatography on DEAE-cellulose; (e) re-chromatography on CM-cellulose; (f) gel filtration on Bio-Gel P-2; (g) partition chromatography on high-pressure liquid chromatography. The homogeneity of the isolated tryptensin was confirmed by thin-layer chromatography and thin-layer electrophoresis. The amino acid analysis of the hydrolysate suggested the following proportional composition: Asp, 1; Val, 1; Ile, 1; Tyr, 1; Phe, 1; His, 1; Arg, 1; Pro, 1. This composition is identical with that of human angiotensin.

scholarly journals Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor

2021 ◽  
Vol 22 (12) ◽  
pp. 6361
Author(s):  
Eunyoung Lee ◽  
Michelle Novais de Paula ◽  
Sangki Baek ◽  
Huynh Kim Khanh Ta ◽  
Minh Tan Nguyen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ion Exchange ◽  
Stem Cell Factor ◽  
Transmembrane Domain ◽  
Coli Strain ◽  
Exchange Chromatography ◽  
Soluble Form ◽  
Human Stem Cell ◽  
E Coli ◽  
Human Stem Cell Factor

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b’a’ domain (PDIb’a’) tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

Strong cation-exchange chromatography of proteins on a sulfoalkylated monolithic cryogel

2015 ◽  
Vol 1386 ◽  
pp. 13-21 ◽  
Author(s):  
Işık Perçin ◽  
Rushd Khalaf ◽  
Bastian Brand ◽  
Massimo Morbidelli ◽  
Orhan Gezici
Keyword(s):  
Cation Exchange ◽  
Exchange Chromatography ◽  
Cation Exchange Chromatography ◽  
Strong Cation Exchange ◽  
Strong Cation Exchange Chromatography
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