scholarly journals Purification, cDNA cloning and expression of 15-oxoprostaglandin 13-reductase from pig lung

1998 ◽  
Vol 330 (1) ◽  
pp. 103-108 ◽  
Author(s):  
Charles Mark ENSOR ◽  
Hongxing ZHANG ◽  
Hsin-Hsiung TAI
Keyword(s):  
Molecular Mass ◽  
Specific Activity ◽  
Leukotriene B4 ◽  
Cloning And Expression ◽  
Library Screening ◽  
Amino Acid Residues ◽  
Calculated Molecular Mass ◽  
Apparent Homogeneity ◽  
Pcr Product ◽  
Cloned Cdna

15-Oxoprostaglandin 13-reductase (PGR) has been purified to apparent homogeneity from pig lung. The enzyme was estimated to have a molecular mass of 36 kDa by both SDS/PAGE and non-denaturing PAGE, indicating that the enzyme is a monomer. 15-Oxo-PGE1, 15-oxo-PGE2 and 15-oxo-PGF2α were found to be substrates for the enzyme, whereas the corresponding 15-hydroxyprostaglandins were not. The reverse reaction, the oxidation of 13,14-dihydro-15-oxo-PGE1 to 15-oxo-PGE1, was not observed. Either NADH or NADPH could serve as a coenzyme. However, the Vmax with NADH was approx. 3-fold that with NADPH, while the Km for NADPH was approx. one-tenth that for NADH. Cloning of the cDNA was achieved by PCR and library screening. A 600 bp PCR product containing the sequences of three different tryptic peptides derived from purified PGR was used for cDNA library screening by plaque hybridization. A cDNA clone that contained the entire PGR coding sequence of 987 bp was obtained. The sequence codes for a protein of 329 amino acid residues with a calculated molecular mass of 35791 Da. Homology analysis indicated that the sequence is virtually identical with that of leukotriene B4 (LTB4) 12-hydroxydehydrogenase [Yokomizo, Ogawa, Uozumi, Kume, Izumi and Shimizu (1996) J. Biol. Chem. 271, 2844-2850]. Expression of this cDNA in Escherichia coli resulted in a protein exhibiting both PGR and LTB4 12-hydroxydehydrogenase activities. However, the specific activity of PGR with 15-oxo-PGE1 as a substrate was approx. 300-fold that of LTB4 12-hydroxydehydrogenase. These results indicate that the cloned cDNA codes for a protein with two different enzyme activities, with 15-oxoprostaglandins as the preferred substrates.

Molecular Cloning and Expression of β-1,4-endoxylanase Gene from Chaetomium cupreum in Yeast Saccharomyces cerevisiae

2013 ◽  
Vol 779-780 ◽  
pp. 191-194
Author(s):  
Hai Yan Zhang ◽  
Wen Rao Li ◽  
Min Li
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Nucleotide Sequence ◽  
Heterologous Expression ◽  
Molecular Mass ◽  
Molecular Cloning ◽  
Cloning And Expression ◽  
Cdna Fragment ◽  
Gene Encoding ◽  
Calculated Molecular Mass

The gene encoding an endo-β-1,4-xylanase (XynCC) fromchaetomium cupreumwas amplified using PCR. The nucleotide sequence of a 690 bp cDNA fragment was determined. Based on the nucleotide sequence, calculated molecular mass of the enzyme was 24.7 kDa. The XynCC gene was inserted into the pYES2 vector and transferred into the cells ofS. cerevisiaeH158 for heterologous expression.

scholarly journals Purification and characterization of a plasma membrane ferricyanide-utilizing NADH dehydrogenase from Ehrlich tumour cells

1996 ◽  
Vol 314 (2) ◽  
pp. 587-593 ◽  
Author(s):  
Antonio del CASTILLO-OLIVARES ◽  
Miguel A. MEDINA ◽  
Ignacio NÚÑEZ de CASTRO ◽  
Javier MÁRQUEZ
Keyword(s):  
Plasma Membrane ◽  
Molecular Mass ◽  
Nadh Dehydrogenase ◽  
Specific Activity ◽  
Gel Filtration ◽  
Maximum Activity ◽  
Exchange Chromatography ◽  
Tumour Cells ◽  
Ammonium Sulphate Precipitation ◽  
Apparent Homogeneity

A ferricyanide-utilizing NADH dehydrogenase (NADH-ferricyanide oxidoreductase) from the plasma membrane of Ehrlich ascites tumour cells has been purified about 1500-fold to apparent homogeneity. The method comprises the isolation of an enriched plasma membrane fraction, solubilization with Triton X-100, ion-exchange chromatography, ammonium sulphate precipitation, Cibacron Blue chromatography and fast-protein liquid chromatography with a Superose-6 gel filtration column. The specific activity of the final pool was more than 61 units/mg protein. The pure enzyme examined by SDS/PAGE displayed only one type of subunit with an apparent molecular mass of 32.0 kDa. The molecular mass of the native protein (117.0 kDa) was estimated by gel filtration; these results suggest a protein composed of four subunits of identical molecular mass. The enzyme was stable in the pH interval between 6 and 9, with maximum activity at pH values from 7.5 to 8.5. The purified enzyme showed Michaelis–Menten kinetics for the substrates, with apparent Km values of 4.3×10-5 M and 6.7×10-5 M for NADH and ferricyanide respectively. The isolated protein was strongly inhibited by Zn2+ and the thiol-specific reagents mersalyl and p-chloromercuribenzenesulphonic acid.

scholarly journals Cloning and expression of the gene for the active PPi-dependent phosphofructokinase of Entamoeba histolytica

1998 ◽  
Vol 329 (3) ◽  
pp. 659-664 ◽  
Author(s):  
Zhihong DENG ◽  
Min HUANG ◽  
Kuber SINGH ◽  
A. Richard ALBACH ◽  
P. Steven LATSHAW ◽  
...  
Keyword(s):  
Entamoeba Histolytica ◽  
Specific Activity ◽  
Ph Dependence ◽  
Cloning And Expression ◽  
Kinetic Properties ◽  
Partial Amino Acid Sequence ◽  
Sequence Identity ◽  
Cloned Cdna ◽  
Cloned Gene ◽  
Substrate Affinities

Pyrophosphate-dependent phosphofructokinase (PPi-PFK) from Entamoeba histolytica (HM-1) was purified from trophozoites. Oligonucleotide probes based on partial amino acid sequence were used to clone and sequence the gene and the cDNA of the enzyme. The molecular mass of the subunit was greater than, and the derived sequence significantly different from, that of the product of the PPi-PFK gene previously cloned from E. histolytica [Huang, Albach, Chang, Tripathi and Kemp (1995) Biochim. Biophys. Acta 1260, 215-217; Bruchhaus, Jacobs, Denart and Tannich (1996) Biochem. J. 316, 57-63]. The sequence identity between the two proteins was 17%. The sequence bore greater identity with the more phylogenetically advanced plant PPi-PFKs than with bacterial PPi-PFKs. The cloned cDNA was expressed and the protein purified. The kinetic properties were identical with those of the enzyme isolated from the organism. Furthermore, the specific activity was more than three orders of magnitude higher than that described for the product of the previously cloned E. histolytica PFK gene [Bruchhaus et al. (1996)]. The pH-dependence and apparent substrate affinities of the cloned enzyme were identical with those of the PPi-PFK in trophozoite extracts, indicating that the product of the cloned gene accounts for most if not all of the PFK activity in E. histolytica trophozoites.

scholarly journals Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase

1994 ◽  
Vol 300 (1) ◽  
pp. 85-90 ◽  
Author(s):  
B Verjans ◽  
F De Smedt ◽  
R Lecocq ◽  
V Vanweyenberg ◽  
C Moreau ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mass ◽  
Bovine Brain ◽  
Cloning And Expression ◽  
Major Protein ◽  
Type I ◽  
Degenerate Primers ◽  
Calculated Molecular Mass ◽  
Major Protein Band ◽  
Inositol 1,4,5 Trisphosphate

In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3. This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE. This material was used to determine internal microsequences. A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE). A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library. The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da. Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein. When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively. Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme. Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme.

scholarly journals Identification, Cloning, and Expression of L-Amino Acid Oxidase from MarinePseudoalteromonassp. B3

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Zhiliang Yu ◽  
Ning Zhou ◽  
Hua Qiao ◽  
Juanping Qiu
Keyword(s):  
Amino Acid ◽  
Cloning And Expression ◽  
Expression Level ◽  
Sequence Information ◽  
Acid Oxidase ◽  
Amino Acid Residues ◽  
Amino Acid Oxidase ◽  
Calculated Molecular Mass ◽  
E Coli ◽  
Sea Area

L-amino acid oxidase (LAAO) is attracting more attentions due to its broad and important biological functions. Recently, an LAAO-producing marine microorganism (strain B3) was isolated from the intertidal zone of Dinghai sea area, China. Physiological, biochemical, and molecular identifications together with phylogenetic analysis congruously suggested that it belonged to the genusPseudoalteromonas. Therefore, it was designated asPseudoalteromonassp. B3. Its capability of LAAO production was crossly confirmed by measuring the products of H2O2, a-keto acids, andNH4+in oxidization reaction. Two rounds of PCR were performed to gain the entire B3-LAAO gene sequence of 1608 bps in length encoding for 535 amino acid residues. This deduced amino acid sequence showed 60 kDa of the calculated molecular mass, supporting the SDS-PAGE result. Like most of flavoproteins, B3-LAAO also contained two conserved typical motifs, GG-motif andβαβ-dinucleotide-binding domain motif. On the other hand, its unique substrate spectra and sequence information suggested that B3-LAAO was a novel LAAO. Our results revealed that it could be functionally expressed inE. coliBL21(DE3) using vectors, pET28b(+) and pET20b(+). However, compared with the native LAAO, the expression level of the recombinant one was relatively low, most probably due to the formation of inclusion bodies. Several solutions are currently being conducted in our lab to increase its expression level.

scholarly journals Enhancement of insect antifreeze protein activity by solutes of low molecular mass.

1998 ◽  
Vol 201 (15) ◽  
pp. 2243-2251 ◽  
Author(s):  
N Li ◽  
C A Andorfer ◽  
J G Duman
Keyword(s):  
Molecular Mass ◽  
Thermal Hysteresis ◽  
Specific Activity ◽  
Freezing Point ◽  
Consensus Sequence ◽  
Ammonium Bicarbonate ◽  
Amino Acid Residues ◽  
Protein Activity ◽  
Dendroides Canadensis ◽  
Thermal Hysteresis Activity

Antifreeze proteins (AFPs) lower the non-equilibrium freezing point of water (in the presence of ice) below the melting point, thereby producing a difference between the freezing and melting points that has been termed thermal hysteresis. In general, the magnitude of the thermal hysteresis depends upon the specific activity and concentration of the AFP. This study describes several low-molecular-mass solutes that enhance the thermal hysteresis activity of an AFP from overwintering larvae of the beetle Dendroides canadensis. The most active of these is citrate, which increases the thermal hysteresis nearly sixfold from 1.2 degrees C in its absence to 6.8 degrees C. Solutes which increase activity approximately fourfold are succinate, malate, aspartate, glutamate and ammonium sulfate. Glycerol, sorbitol, alanine and ammonium bicarbonate increased thermal hysteresis approximately threefold. Interestingly, 0.5 mol l-1 sodium sulfate eliminated activity. Solute concentrations between 0.25 and 1 mol l-1 were generally required to elicit optimal thermal hysteresis activity. Glycerol is the only one of these enhancing solutes that is known to be present at these concentrations in overwintering D. canadensis, and therefore the physiological significance of most of these enhancers is unknown. The mechanism(s) of this enhancement is also unknown. The AFP used in this study (DAFP-4) is nearly identical to previously described D. canadensis AFPs. The mature protein consists of 71 amino acid residues arranged in six 12- or 13-mer repeats with a consensus sequence consisting of Cys-Thr-X3-Ser-X5-X6-Cys-X8-X9-Ala-X11-Thr-X1 3, where X3 and X11 tend to be charged residues, X5 tends to be Thr or Ser, X6 to be Asn or Asp, X9 to be Asn or Lys and X13 to be Ala in the 13-mers. DAFP-4 is shorter by one repeat than previously described D. canadensis AFPs.

Thermus aquaticus ATCC 33923 Amylomaltase Gene Cloning and Expression and Enzyme Characterization: Production of Cycloamylose

1999 ◽  
Vol 65 (3) ◽  
pp. 910-915 ◽  
Author(s):  
Yoshinobu Terada ◽  
Kazutoshi Fujii ◽  
Takeshi Takaha ◽  
Shigetaka Okada
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Molecular Mass ◽  
Minimum Degree ◽  
Cloning And Expression ◽  
Gene Cloning And Expression ◽  
Thermus Aquaticus ◽  
Average Molecular Mass ◽  
Calculated Molecular Mass ◽  
E Coli

ABSTRACT The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of theEscherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75°C in a 10-min reaction with maltotriose and was stable at temperatures up to 85°C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic α-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coliamylomaltase and the plant D-enzyme.

scholarly journals Enzymatic Properties of a Novel Liquefying α-Amylase from an Alkaliphilic Bacillus Isolate and Entire Nucleotide and Amino Acid Sequences

1998 ◽  
Vol 64 (9) ◽  
pp. 3282-3289 ◽  
Author(s):  
Kazuaki Igarashi ◽  
Yuji Hatada ◽  
Hiroshi Hagihara ◽  
Katsuhisa Saeki ◽  
Mikio Takaiwa ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Molecular Mass ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Maximum Activity ◽  
Amino Acid Sequences ◽  
Ph Optimum ◽  
Calculated Molecular Mass

ABSTRACT A novel liquefying α-amylase (LAMY) was found in cultures of an alkaliphilic Bacillus isolate, KSM-1378. The specific activity of purified LAMY was approximately 5,000 U mg of protein−1, a value two- to fivefold greater between pH 5 and 10 than that of an industrial, thermostable Bacillus licheniformis enzyme. The enzyme had a pH optimum of 8.0 to 8.5 and displayed maximum activity at 55°C. The molecular mass deduced from sodium dodecyl sulfate-polyacrylamide gel electrophoresis was approximately 53 kDa, and the apparent isoelectric point was around pH 9. This enzyme efficiently hydrolyzed various carbohydrates to yield maltotriose, maltopentaose, maltohexaose, and maltose as major end products after completion of the reaction. Maltooligosaccharides in the maltose-to-maltopentaose range were unhydrolyzable by the enzyme. The structural gene for LAMY contained a single open reading frame 1,548 bp in length, corresponding to 516 amino acids that included a signal peptide of 31 amino acids. The calculated molecular mass of the extracellular mature enzyme was 55,391 Da. LAMY exhibited relatively low amino acid identity to other liquefying amylases, such as the enzymes from B. licheniformis (68.9%), Bacillus amyloliquefaciens (66.7%), and Bacillus stearothermophilus (68.6%). The four conserved regions, designated I, II, III, and IV, and the putative catalytic triad were found in the deduced amino acid sequence of LAMY. Essentially, the sequence of LAMY was consistent with the tertiary structures of reported amylolytic enzymes, which are composed of domains A, B, and C and which include the well-known (α/β)8 barrel motif in domain A.

Cloning, Expression, and Characterization of Mouse Tissue Factor Pathway Inhibitor (TFPI)

1998 ◽  
Vol 79 (02) ◽  
pp. 306-309 ◽  
Author(s):  
Dougald Monroe ◽  
Julie Oliver ◽  
Darla Liles ◽  
Harold Roberts ◽  
Jen-Yea Chang
Keyword(s):  
Amino Acid ◽  
Tissue Factor ◽  
Signal Peptide ◽  
Tissue Factor Pathway Inhibitor ◽  
Factor Xa ◽  
Protein Sequences ◽  
Cloning And Expression ◽  
Mouse Tissue ◽  
Amino Acid Residues ◽  
Tissue Factor Pathway

SummaryTissue factor pathway inhibitor (TFPI) acts to regulate the initiation of coagulation by first inhibiting factor Xa. The complex of factor Xa/ TFPI then inhibits the factor VIIa/tissue factor complex. The cDNA sequences of TFPI from several different species have been previously reported. A high level of similarity is present among TFPIs at the molecular level (DNA and protein sequences) as well as in biochemical function (inhibition of factor Xa, VIIa/tissue factor). In this report, we used a PCR-based screening method to clone cDNA for full length TFPI from a mouse macrophage cDNA library. Both cDNA and predicted protein sequences show significant homology to the other reported TFPI sequences, especially to that of rat. Mouse TFPI has a signal peptide of 28 amino acid residues followed by the mature protein (in which the signal peptide is removed) which has 278 amino acid residues. Mouse TFPI, like that of other species, consists of three tandem Kunitz type domains. Recombinant mouse TFPI was expressed in the human kidney cell line 293 and purified for functional assays. When using human clotting factors to investigate the inhibition spectrum of mouse TFPI, it was shown that, in addition to human factor Xa, mouse TFPI inhibits human factors VIIa, IXa, as well as factor XIa. Cloning and expression of the mouse TFPI gene will offer useful information and material for coagulation studies performed in a mouse model system.

Binding of 3-phosphoglycerate leads to both activation and stabilisation of ADP-glucose pyrophosphorylase from apple leaves

2005 ◽  
Vol 32 (9) ◽  
pp. 839
Author(s):  
Rui Zhou ◽  
Lailiang Cheng
Keyword(s):  
Heat Treatment ◽  
Thermal Stability ◽  
Enzyme Activity ◽  
Inorganic Phosphate ◽  
Thermal Inactivation ◽  
Specific Activity ◽  
Apple Leaves ◽  
Apparent Homogeneity ◽  
Adp Glucose Pyrophosphorylase ◽  
High Concentrations

Apple leaf ADP-glucose pyrophosphorylase was purified 1436-fold to apparent homogeneity with a specific activity of 58.9 units mg–1. The enzyme was activated by 3-phosphoglycerate (PGA) and inhibited by inorganic phosphate (Pi) in the ADPG synthesis direction. In the pyrophosphorolytic direction, however, high concentrations of PGA (> 2.5 mm) inhibited the enzyme activity. The enzyme was resistant to thermal inactivation with a T0.5 (temperature at which 50% of the enzyme activity is lost after 5 min incubation) of 52°C. Incubation with 2 mm PGA or 2 mm Pi increased T0.5 to 68°C. Incubation with 2 mm dithiothreitol (DTT) decreased T0.5 to 42°C, whereas inclusion of 2 mm PGA in the DTT incubation maintained T0.5 at 52°C. DTT-induced decrease in thermal stability was accompanied by monomerisation of the small subunits. Presence of PGA in the DTT incubation did not alter the monomerisation of the small subunits of the enzyme induced by DTT. These findings indicate that binding of PGA renders apple leaf AGPase with a conformation that is not only more efficient in catalysis but also more stable to heat treatment. The physiological significance of the protective effect of PGA on thermal inactivation of AGPase is discussed.

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