A full-length cDNA encoding the pig cytosolic aspartate aminotransferase (EC 18.104.22.168) (cAspAT) was constructed from two overlapping cDNA clones. One clone (Lm pcAAT-8) isolated from a λ gt 10 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 λ gt 10 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 ± 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: Alal (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.
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