Cloning, sequencing and functional expression of a DNA encoding pig cytosolic malate dehydrogenase: purification and characterization of the recombinant enzyme.
Using the polymerase chain reaction, DNA encoding cytosolic malate dehydrogenase (cMDH) has been cloned from a pig heart cDNA library. Large amounts of the enzyme (30 mg per litre of original culture) have been produced in Escherichia coli using an inducible expression vector (pKK223-3) in which the 5'-non-coding region of the gene was replaced with the tac promoter. The complete nucleotide sequence of the DNA is reported for the first time. The recombinant cMDH purified was shown to be identical to the native enzyme according to: chromatographic behaviour, isoelectric point, N-terminal amino acid sequence, and physiochemical and catalytic properties.
Trejo
F
F
Departament de Bioquímica i Biologia Molecular, Facultat de Química, Universitat de Barcelona, Spain.
Costa
M
M
Gelpí
J L
JL
Busquets
M
M
Clarke
A R
AR
Holbrook
J J
JJ
Cortés
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology; D004247:DNA; D018076:DNA, Complementary; D004926:Escherichia coli; D008291:Malate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D013552:Swine
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D004247:DNA; D008291:Malate Dehydrogenase