cDNA cloning and expression of pig cytosolic aspartate aminotransferase in Escherichia coli: amino-terminal heterogeneity of expressed products and lack of its correlation with enzyme function.
A full-length cDNA encoding the pig cytosolic aspartate aminotransferase (EC 2.6.1.1) (cAspAT) was constructed from two overlapping cDNA clones. One clone (Lm pcAAT-8) isolated from a lambda gt10 pig heart cDNA library contained a 3' untranslated sequence, a poly(A) segment, and a part of the coding region for amino acid positions 127-412. Another clone (Lm pcAAT-107) isolated from a lambda gt10 primer extension library contained the coding region for amino acid positions 1-148 and a 5' untranslated sequence. Rejoining of the cDNA inserts of the two clones and recloning into pUC18 gave rise to a cDNA covering an entire coding sequence for pig cAspAT mRNA. Insertion into pKK223-3 yielded an expression plasmid, ppcAAT200. Escherichia coli JM105 cells transfected with ppcAAT200 overproduced pig cAspAT to an extent of about 3% of the total cellular soluble proteins. The expressed product was indistinguishable from the alpha subform of cAspAT isolated from pig heart in terms of specific activity, absorption spectra, molecular size, crystalline form, and immunological reactivity with anti pig cAspAT antibody. Compared with the amino-terminal sequence (Ala-Pro-Pro-) reported for pig heart cAspAT, the recombinant pig cAspAT showed heterogeneity in the amino-terminal sequence: Ala 1 (26%), Pro2 (54%), and Pro3 (19%). Construction of a mutant cAspAT with deletion of residues 1-3 and its comparison with the wild-type enzyme revealed that loss of the three amino-terminal residues does not affect the catalytic activity and structural integrity of the enzyme.
Nagashima
F
F
Department of Biochemistry, Kumamoto University Medical School, Japan.
Tanase
S
S
Fukumoto
Y
Y
Joh
T
T
Nomiyama
H
H
Tsuzuki
T
T
Shimada
K
K
Kuramitsu
S
S
Kagamiyama
H
H
Morino
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001219:Aspartate Aminotransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D003600:Cytosol / Q000201:enzymology; D004247:DNA / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013552:Swine
D003062:Codon; D011994:Recombinant Proteins; D004247:DNA; D001219:Aspartate Aminotransferases