期刊
PROTEIN EXPRESSION AND PURIFICATION
卷 57, 期 2, 页码 116-126出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.pep.2007.10.016
关键词
tryptophan hydroxylase; expression; purification; catalytic domain; kinetic; crystallization
Tryptophan hydroxylase (TPH) [EC 1.14.16.4] catalyzes the conversion of tryptophan to 5-hydroxytryptophan, which is the first and rate-determining step in the biosynthesis of the neurotransmitter serotonin. We have expressed the catalytic domain of chicken (Gallus gallus) TPH isoform 1 in Escherichia coli in high yield. The enzyme was highly purified using only one anion exchange and one gel filtration, with a yield of 11 mg/L culture and a specific activity of 0.60 mu mol/min/mg. The K-m values were determined to K-m,K-tryptophan = 7.7 +/- 0.7 mu M, K-m,K-BH4 = 324 +/- 10 mu M and K-m,K-O2 = 39 +/- 2 mu M. substrate inhibition by tryptophan was observed at concentrations above 15 mu M. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3 angstrom resolution has been collected. (C) 2007 Elsevier Inc. All rights reserved.
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