Cloning and expression of Saccharomyces cerevisiae SUC2 gene in yeast platform and characterization of recombinant enzyme biochemical properties

Inositol monophosphatase (EC 3.1.3.25) is a key enzyme in the phosphoinositide cell-signalling system. Its role is to provide inositol required for the resynthesis of phosphatidylinositol and polyphosphoinositides. It is the probable pharmacological target for lithium action in brain. Using probes derived from the bovine inositol monophosphatase cDNA we have isolated cDNA clones encoding the human and rat brain enzymes. The enzyme is highly conserved in all three species (79% identical). The coding region of the human cDNA was inserted into a bacterial expression vector. The expressed recombinant enzyme was purified and its biochemical properties examined. The human enzyme is very similar to the bovine enzyme.

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Cloning and expression of Saccharomyces cerevisiae copper-metallothionein gene in Escherichia coli and characterization of the recombinant protein

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1993.tb17689.x ◽

1993 ◽

Vol 212 (2) ◽

pp. 521-528 ◽

Cited By ~ 16

Author(s):

Zehra SAYERS ◽

Patricia BROUILLON ◽

Constantin E. VORGIAS ◽

Hans F. NOLTING ◽

Christoph HERMES ◽

...

Keyword(s):

Escherichia Coli ◽

Saccharomyces Cerevisiae ◽

Recombinant Protein ◽

Cloning And Expression ◽

Metallothionein Gene

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Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme

Biotechnology Research International ◽

10.1155/2012/951267 ◽

2012 ◽

Vol 2012 ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Dominic W. S. Wong ◽

Victor J. Chan ◽

Amanda A. McCormack ◽

Ján Hirsch ◽

Peter Biely

Keyword(s):

Glucuronic Acid ◽

Active Form ◽

Schizophyllum Commune ◽

Constitutive Expression ◽

Recombinant Enzyme ◽

Cloning And Expression ◽

Gene Encoding ◽

Glucuronoyl Esterase ◽

Esterase Gene

The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were Km 0.25 mM, Vmax 16.3 μM⋅min−1, and kcat 9.27 s−1 with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-β-D-xylopyranoside as the substrate.

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Characterization of the intron-containing citrate synthase gene from the alkanotrophic yeast Candida tropicalis : cloning and expression in Saccharomyces cerevisiae

Archives of Microbiology ◽

10.1007/s002030050463 ◽

1997 ◽

Vol 168 (1) ◽

pp. 8-15 ◽

Cited By ~ 4

Author(s):

Mitsuyoshi Ueda ◽

Shin-ichi Sanuki ◽

Hiroyuki Kawachi ◽

Kaori Shimizu ◽

Haruyuki Atomi ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Candida Tropicalis ◽

Citrate Synthase ◽

Cloning And Expression

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Molecular Cloning and Expression of an Endo-β-1,4-D-glucanase I (Avicelase I) Gene from Bacillus cellulyticus K-12 and Characterization of the Recombinant Enzyme

Applied Biochemistry and Biotechnology ◽

10.1385/abab:80:2:121 ◽

1999 ◽

Vol 80 (2) ◽

pp. 121-140 ◽

Cited By ~ 9

Author(s):

Tae-Kyun Lee ◽

Cheorl-Ho Kim

Keyword(s):

Molecular Cloning ◽

Recombinant Enzyme ◽

Cloning And Expression ◽

K 12 ◽

I Gene

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Cystathionine γ-synthase from Arabidopsis thaliana: purification and biochemical characterization of the recombinant enzyme overexpressed in Escherichia coli

Biochemical Journal ◽

10.1042/bj3310639 ◽

1998 ◽

Vol 331 (2) ◽

pp. 639-648 ◽

Cited By ~ 57

Author(s):

Stéphane RAVANEL ◽

Bertrand GAKIÈRE ◽

Dominique JOB ◽

Roland DOUCE

Keyword(s):

Biochemical Characterization ◽

Biochemical Properties ◽

Recombinant Enzyme ◽

Enzyme Inactivation ◽

Replacement Reaction ◽

Protein Precursor ◽

Long Wavelength ◽

Ping Pong ◽

Km Value

Cystathionine γ-synthase catalyses the first reaction specific for methionine biosynthesis in plants, the γ-replacement of the phosphoryl substituent of O-phosphohomoserine by cysteine. A cDNA encoding cystathionine γ-synthase from Arabidopsis thalianahas been cloned and used to overexpress the enzyme in Escherichia coli.The native recombinant enzyme is a homotetramer composed of 53 kDa subunits, each being tightly associated with one molecule of pyridoxal 5´-phosphate that binds at lysine-379 of the protein precursor. The replacement reaction follows a Ping Pong mechanism with a Vmax of 33.6 units/mg and Km values of 2.5 mM and 460 µM for O-phosphohomoserine and cysteine respectively. The protective effect of O-phosphohomoserine against enzyme inactivation by propargylglycine indicated that the Kd for the substrate is approx. 1/2500 of its Km value. Thus most of these biochemical properties are similar to those previously reported for plant and bacterial cystathionine γ-synthases. However, the plant enzyme differs markedly from its enterobacterial counterparts because it catalyses a very faint γ-elimination of O-phosphohomoserine in the absence of cysteine, this process being about 1/2700 as fast as the γ-replacement reaction and approx. 1/1500 as fast as the γ-elimination catalysed by the E. colienzyme. This huge difference could be attributed to the inability of the A. thalianacystathionine γ-synthase to accumulate a long-wavelength-absorbing species that is characteristic for the efficient γ-elimination reaction catalysed by the enterobacterial enzyme.

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