Dataset Information

High-level expression and purification of rat monoamine oxidase A (MAO A) in Pichia pastoris: comparison with human MAO A.

ABSTRACT: The high-level heterologous expression in Pichia pastoris, purification and characterization of recombinant membrane-bound rat liver monoamine oxidase A (MAO A) are described. A 1-L culture of cells produces approximately 700 U of rat MAO A activity. The rat MAO A activity is found in outer mitochondrial membrane of the cell. Using a modification of the human MAO A purification procedure, approximately 200mg of recombinant rat MAO A is purified in a 43% yield and exhibits a molecular weight of approximately 60,000 kDa on SDS-PAGE. The purified enzyme contains a covalently bound FAD and forms a N(5) flavocyanine adduct on inhibition by clorgyline. Edman sequencing shows that the amino terminus of rat MAO A is blocked at an N-terminal threonyl residue. The purified rat enzyme exhibits a higher thermal stability than does purified human MAO A. Compared with human MAO A, rat MAO A oxidizes serotonin or kynuramine with twofold higher k(cat)/K(m) values, oxidizes phenethylamine with a 6.7-fold higher catalytic efficiency and benzylamine with a approximately 40-fold higher catalytic efficiency. Although approximately 90% identical in sequence to human MAO A, rat MAO A is a more efficient catalyst for amine neurotransmitter oxidation.

SUBMITTER: Wang J

PROVIDER: S-EPMC2827670 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1–Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (Kd for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.

Dataset Information

High-level expression and purification of rat monoamine oxidase A (MAO A) in Pichia pastoris: comparison with human MAO A.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets