Journal article
Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli
Protein expression and purification, Vol.84(2), pp.219-223
08/2012
DOI: 10.1016/j.pep.2012.05.007
PMCID: PMC3525113
PMID: 22659380
Abstract
► We produce enzymatically active recombinant human tyrosine hydroxylase (hTH) in E. coli. ► The protein is expressed as a maltose-binding protein fusion and cleaved after affinity capture. ► We report the first purification resulting in cleaved, active hTH in high yield.
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 ∼23mg of pure, active and stable enzyme exhibiting a specific activity of ∼17nmol/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.
Details
- Title: Subtitle
- Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli
- Creators
- Colin A Higgins - Division of Medicinal and Natural Products Chemistry, The University of Iowa College of Pharmacy, 115 S. Grand Ave., Iowa City, IA 52242, USALydia M Vermeer - Division of Medicinal and Natural Products Chemistry, The University of Iowa College of Pharmacy, 115 S. Grand Ave., Iowa City, IA 52242, USAJonathan A Doorn - Division of Medicinal and Natural Products Chemistry, The University of Iowa College of Pharmacy, 115 S. Grand Ave., Iowa City, IA 52242, USADavid L Roman - Division of Medicinal and Natural Products Chemistry, The University of Iowa College of Pharmacy, 115 S. Grand Ave., Iowa City, IA 52242, USA
- Resource Type
- Journal article
- Publication Details
- Protein expression and purification, Vol.84(2), pp.219-223
- DOI
- 10.1016/j.pep.2012.05.007
- PMID
- 22659380
- PMCID
- PMC3525113
- NLM abbreviation
- Protein Expr Purif
- ISSN
- 1046-5928
- eISSN
- 1096-0279
- Publisher
- Elsevier Inc
- Language
- English
- Date published
- 08/2012
- Academic Unit
- Pharmacy; Iowa Neuroscience Institute; Pharmaceutical Sciences and Experimental Therapeutics; Medicinal and Natural Products Chemistry
- Record Identifier
- 9984065384202771
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