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

被引:21
作者
TIPPER, JP [1 ]
CITRON, BA [1 ]
RIBEIRO, P [1 ]
KAUFMAN, S [1 ]
机构
[1] NIMH, NEUROCHEM LAB, BETHESDA, MD 20892 USA
关键词
TRYPTOPHAN HYDROXYLASE; RABBIT BRAIN; HUMAN BRAIN; MOLECULAR CLONING; EXPRESSION;
D O I
10.1006/abbi.1994.1523
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 hyman 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. (C) 1994 Academic Press, Inc.
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页码:445 / 453
页数:9
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