CLONING AND EXPRESSION OF RABBIT AND HUMAN BRAIN TRYPTOPHAN-HYDROXYLASE CDNA IN ESCHERICHIA-COLI

Rabbit and human brain tryptophan hydroxylase were cloned and expresse d in Escherichia coli. Each of the respective cDNAs, including the com plete coding sequence of tryptophan hydroxylase, was obtained by rever se transcription of rabbit or human brain mRNA and subcloned into the expression vector pET-3C. The expressed rabbit brain tryptophan hydrox ylase activity, measured in the presence of tetrahydrobiopterin, repre sents approximately a 50-fold enhancement in yield (units/g tissue (we t wt) over that of a rabbit brain extract. Likewise, the level of expr essed human brain tryptophan hydroxylase is approximately 57 times the average yield previously reported for a human brain homogenate and ap proximately 10-times the activity of homogenates of hyman raphe nucleu s. The rabbit brain and pineal-derived tryptophan hydroxylase sequence s 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 expr essed enzymes were studied and compared with those of native tryptopha n hydroxylases. (C) 1994 Academic Press, Inc.