Expression of mammalian tyrosine aminotransferase in Saccharomyces cerevisiae and Escherichia coli. Purification to homogeneity and characterization of the enzyme overproduced in the bacteria.
Rat liver tyrosine aminotransferase has been expressed in Saccharomyces cerevisiae and Escherichia coli. In yeast, the extent of production is 20-fold higher than that in rat liver after induction by dexamethasone, and reaches 250-fold higher in an E. coli strain carrying the T7 RNA polymerase transcription system. About 250 mg pure and homogeneous enzyme was obtained from 50 g transformed E. coli cells. Determination of Mr and pI, as well as analysis of N- and C-terminal amino acids, suggest that the isolated protein is native. The catalytic properties, similar to those of the enzyme from rat liver, confirm that it is fully active and that post-translational modifications in the mammalian cells are not essential for activity. Pyridoxal 5'-phosphate strongly protects the enzyme against thermal inactivation. After denaturation, 10 thiol groups, out of 16 in the polypeptide chain, react with 5,5'-dithiobis(2-nitrobenzoic acid) whereas only five or six are accessible under native conditions. Two thiols are rapidly modified with concomitant inactivation of the apoenzyme, but pyridoxal 5'-phosphate partially protects them in the holoenzyme. The results are interpreted in the light of the structure/function relationship in this enzyme.
Dietrich
J B
JB
Laboratoire de Biochimie, Centre National de la Recherche Scientifique, Strasbourg, France.
Lorber
B
B
Kern
D
D
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D002384:Catalysis; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003854:Deoxyribonucleotides; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D006358:Hot Temperature; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D010957:Plasmids; D011487:Protein Conformation; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014444:Tyrosine Transaminase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D003854:Deoxyribonucleotides; D014444:Tyrosine Transaminase