A simple two step procedure for purification of the catalytic domain of chicken tryptophan hydroxylase 1 in a form suitable for crystallization

Protein Expr Purif. 2008 Feb;57(2):116-26. doi: 10.1016/j.pep.2007.10.016. Epub 2007 Nov 20.

Abstract

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 micromol/min/mg. The K(m) values were determined to K(m, tryptophan)=7.7+/-0.7 microM, K(m, BH4)=324+/-10 microM and K(m, O2)=39+/-2 microM. Substrate inhibition by tryptophan was observed at concentrations above 15 microM. Furthermore, the purified enzyme has been crystallized without 7,8-dihydro-L-biopterin and a data set to 3A resolution has been collected.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Biotechnology / methods*
  • Catalytic Domain*
  • Chickens / metabolism*
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • Crystallography, X-Ray
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Sequence Alignment
  • Tryptophan Hydroxylase / chemistry*
  • Tryptophan Hydroxylase / isolation & purification*

Substances

  • Tryptophan Hydroxylase