Cloning and expression of rabbit and human brain tryptophan hydroxylase cDNA in Escherichia coli.
Rabbit and human brain tryptophan hydroxylase were cloned and expressed in Escherichia coli. Each of the respective cDNAs, including the complete coding sequence of tryptophan hydroxylase, was obtained by reverse transcription of rabbit or human brain mRNA and subcloned into the expression vector pET-3C. The expressed rabbit brain tryptophan hydroxylase activity, measured in the presence of tetrahydrobiopterin, represents approximately a 50-fold enhancement in yield (units/g tissue (wet wt) over that of a rabbit brain extract. Likewise, the level of expressed human brain tryptophan hydroxylase is approximately 57 times the average yield previously reported for a human brain homogenate and approximately 10-times the activity of homogenates of human raphe nucleus. The rabbit brain and pineal-derived tryptophan hydroxylase sequences varied by disparities in six amino acid residues (99% identity). The human carcinoid and brain peptide sequences varied by disparities in 18 amino acid residues (96% identity). Several properties of both expressed enzymes were studied and compared with those of native tryptophan hydroxylases.
Tipper
J P
JP
Laboratory of Neurochemistry, National Institute of Mental Health, Bethesda, Maryland 20892.
Citron
B A
BA
Ribeiro
P
P
Kaufman
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004926:Escherichia coli; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014365:Tryptophan Hydroxylase / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D012333:RNA, Messenger; D011994:Recombinant Proteins; D014365:Tryptophan Hydroxylase