Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Aug;84(2):219-23.
doi: 10.1016/j.pep.2012.05.007. Epub 2012 Jun 1.

Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli

Colin A Higgins  1 , Lydia M Vermeer, Jonathan A Doorn, David L Roman
Affiliations

Expression and purification of recombinant human tyrosine hydroxylase as a fusion protein in Escherichia coli

Colin A Higgins et al. Protein Expr Purif. 2012 Aug.

Abstract

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.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
(A) hTH/pMALc2H10T construct used to express recombinant tyrosine hydroxylase. The TH insert size is 1500 bp, and the MBP–TH fusion protein coding sequence is 2703 bp. The BamHI and SalI restriction sites used to clone TH are unique in the vector and insert. (B) The scheme used to purify hTH consisting of two chromatography steps and an enzymatic digestion with TEV protease (purification also provided).
Fig. 2
Fig. 2
Typical gels representing the purification procedure. (A) After the crude sample is run through an amylose resin column and the MBP-bound TH is eluted off using maltose. Lane (1) MW marker, (2) load (crude lysate), (3) amylose column unbound sample flow-through, (4) wash, 5–20: eluted fractions. All fractions were analyzed by SDS–PAGE, and fractions containing the MBP–TH construct were further purified. (B) TH and MBP after cleavage by TEV for 8 h at 4C; TH and MBP are cleaved (56 and 44 kDa, respectively). (C) Post-HiPrep Q FF 16/10 column using a salt gradient for elution. MBP elutes first, followed by TH. (D) Western blot analysis for TH after the final purification step.
Fig. 3
Fig. 3
Activity of tyrosine hydroxylase at each step in the purification, as determined by L-DOPA production. ten micrograms of pure TH was incubated with tyrosine, iron, and cofactor. HPLC analysis determined the production of L-DOPA over a 20 min time course.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Elsworth JD, Roth RH. Dopamine synthesis, uptake, metabolism, and receptors: relevance to gene therapy of Parkinson’s disease. Exp Neurol. 1997;144:4–9. - PubMed
    1. Nagatsu T, Levitt M, Udenfriend S. Tyrosine hydroxylase. The Initial step in norepinephrine biosynthesis. J Biol Chem. 1964;239:2910–2917. - PubMed
    1. Daubner SC, Lauriano C, Haycock JW, Fitzpatrick PF. Site-directed mutagenesis of serine 40 of rat tyrosine hydroxylase. Effects of dopamine and cAMP-dependent phosphorylation on enzyme activity. J Biol Chem. 1992;267:12639–12646. - PubMed
    1. Xu Y, Stokes AH, Roskoski R, Jr, Vrana KE. Dopamine, in the presence of tyrosinase, covalently modifies and inactivates tyrosine hydroxylase. J Neurosci Res. 1998;54:691–697. - PubMed
    1. Mexas LM, Florang VR, Doorn JA. Inhibition and covalent modification of tyrosine hydroxylase by 3,4-dihydroxyphenylacetaldehyde, a toxic dopamine metabolite. Neurotoxicology. 2011;40:471–477. - PMC - PubMed

Publication types

MeSH terms

Substances