Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli.
Tyrosine hydroxylase is the rate-limiting step in the synthesis of dopamine and is tightly regulated. Previous studies have shown it to be covalently modified and potently inhibited by 3,4-dihydroxyphenylacetaldehyde (DOPAL), an endogenous neurotoxin via dopamine catabolism which is relevant to Parkinson's disease. In order to elucidate the mechanism of enzyme inhibition, a source of pure, active tyrosine hydroxylase was necessary. The cloning and novel purification of human recombinant TH from Escherichia coli is described here. This procedure led to the recovery of ~23 mg of pure, active and stable enzyme exhibiting a specific activity of ~17 nmol/min/mg. The enzyme produced with this procedure can be used to delineate the tyrosine hydroxylase inhibition by DOPAL and its relationship to Parkinson's disease. This procedure improves upon previous methods because the fusion protein gives rise to high expression and convenient affinity-capture, and the cleaved and highly purified hTH makes the product useful for a wider variety of applications.
Division of Medicinal and Natural Products Chemistry, The University of Iowa College of Pharmacy, Iowa City, IA 52242, USA. colin-higgins@uiowa.edu
Colin A
CA
Higgins
Lydia M
LM
Vermeer
Jonathan A
JA
Doorn
David L
DL
Roman
D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014446:Tyrosine 3-Monooxygenase