cDNA Cloning and Expression of Pig Cytosolic Aspartate Aminotransferase in Escherichia colt Amino-Terminal Heterogeity of Expressed Products and Lack of Its Correlation with Enzyme Function

Fujio Nagashima, Sumio Tanase, Yuhji Fukumoto, Tadashi Joh, Hisayuki Nomiyama, Teruhisa Tsuzuki, Kazunori Shimada, Seiki Kuramitsu, Hiroyuki Kagamiyama, Yoshimasa Morino

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Abstract

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 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.

Original languageEnglish
Pages (from-to)1153-1160
Number of pages8
JournalBiochemistry
Volume28
Issue number3
DOIs
Publication statusPublished - Jan 1 1989

All Science Journal Classification (ASJC) codes

  • Biochemistry

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    Nagashima, F., Tanase, S., Fukumoto, Y., Joh, T., Nomiyama, H., Tsuzuki, T., Shimada, K., Kuramitsu, S., Kagamiyama, H., & Morino, Y. (1989). cDNA Cloning and Expression of Pig Cytosolic Aspartate Aminotransferase in Escherichia colt Amino-Terminal Heterogeity of Expressed Products and Lack of Its Correlation with Enzyme Function. Biochemistry, 28(3), 1153-1160. https://doi.org/10.1021/bi00429a033