High-Level Expression of Human Liver Monoamine Oxidase B in Pichia pastoris

Newton-Vinson, Paige; Hubálek, František; Edmondson, Dale E.

doi:10.1006/prep.2000.1309

Cited by 108 publications

(97 citation statements)

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“…The catalytic properties of purified recombinant rMAOB is determined using benzylamine as substrate and compared (Table 3) with those reported previously for recombinant human MAOB expressed in P. pastoris [13,24]. The observed K m (benzylamine) of the P. pastoris expressed rMAOB (176 ± 15 µM) is similar to the value reported earlier for rMAOB (140 ± 10 µM) expressed in S. cerevisiae [9].…”

Section: Catalytic Activitysupporting

confidence: 71%

“…Purification of rMAOB was done following the hMAOB protocol [13,19] with some minor modifications to improve the yield and purity of the enzyme. Approximately 130 gram (wet cell weight) of P. pastoris cells (ca.…”

Section: Purification Of Rmaobmentioning

confidence: 99%

“…Liberation of the enzyme from its membrane environment requires hydrolysis of the phospholipids by phospholipase treatment followed by extraction with Triton-X 100 detergent. An improvement in enzyme purification over that previously described for recombinant human MAOB [13] is the use of an one step anion exchange column chromatography using Bio-Rad High-Q resin (also now routinely used for the purification of hMAOB), as described in the Methods section. This method allows reproducible fractionation of the Triton-X 100 extract to obtain the enzyme in a pure form and is preferable to the previously published polymer fractionation procedure.…”

Section: Membrane Localization and Purification Of Rmaobmentioning

confidence: 99%

“…The induction continued for four days by supplementing with methanol after every 24 hours. Aliquots (1 ml) of each culture were removed every 24 hours and rMAOB expression levels measured by measuring benzylamine oxidase activity as reported earlier for hMAOB [13]. The best expression level was observed from a single colony obtained from 1.0mg/ml G-418 sulfate plate and was used for large scale fermentation following the protocols as described below.…”

mentioning

confidence: 99%

“…Although previous studies have been reported on expression of rMAOB in E. coli [8], and in S. cerevisiae [9,10], the levels of functional proteins and the lack of details on the properties of the expressed enzyme from these systems provides an incentive to explore other systems for the high level expression and subsequent purification of rMAOB. The methylotrophic yeast Pichia pastoris has been used for heterologous expression of a number of proteins [11,12] and has been used successfully in our previous work for large scale expression of human monoamine oxidases A and B [13,14]. The availability of these expression systems provided levels of pure functional enzymes to permit subsequent structural studies [15,16].…”

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confidence: 99%

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Characterization of detergent purified recombinant rat liver monoamine oxidase B expressed in Pichia pastoris

Upadhyay

Edmondson

2008

Protein Expression and Purification

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The high level expression and purification of rat monoamine oxidase B (rMAOB) in the methylotrophic yeast Pichia pastoris is reported. Nearly 100 mg of purified rMAOB is obtained from 130 grams (wet weight) of cells (0.5 liter of culture). The MALDI-TOF mass spectrum of the purified protein shows a single species with a molecular mass of 59.228 ± 0.064 kDa, which agrees with the calculated molecular weight of 59.172 kDa for the rMAOB protein sequence assuming one mole of covalent FAD per mole of the enzyme. Consistent with the MALDI-MS data, purified rMAOB shows a single band near 60 kDa in Coomassie stained SDS/PAGE gel as well as on Western blot analyses performed using antiseras raised against human MAOA and BSA conjugated FAD. A partial amino acid sequence of the purified protein is confirmed to be that of the wild type rMAOB by in-gel trypsin digestion and MALDI-TOF-MS analyses of the liberated peptide fragments. Steady state kinetic data show that purified rMAOB exhibits a K m (amine) of 176 ± 15 µM and a k cat of 497 ± 83 min −1 for benzylamine oxidation, and a K m (O 2 ) of 170 ± 10 µM. Kinetic parameters obtained for purified rMAOB are compared with those reported earlier for recombinant human liver MAOB expressed in Pichia pastoris. KeywordsMonoamine Oxidase B; Rat Liver MAOB; Pichia pastoris Monoamine oxidase B (MAOB) 1 is an outer mitochondrial membrane bound flavoenzyme responsible for oxidative deamination and subsequent degradation of dopamine and dietary amines in our body [1]. These oxidation processes are important in maintaining physiological levels of dopamine and free dietary amines in our body and proper functioning of the central nervous system. Age related increases in the expression levels of MAOB in neural tissues [2,3] are implicated in several neurological disorders like, Parkinson's and Alzheimer's diseases and selective MAOB inhibitors are clinically proven drug adjuvants for treating these disorders [4,5].Owing to their therapeutic applications, developments of MAOB inhibitors with greater clinical potency have gained momentum in recent years. Rat, being one of the most common 1 This work was supported by National Institute of Health Grant GM-29433 (DEE).2To whom correspondence should be addressed: 1510 Clifton Road NE, Atlanta, GA 30322. E-mail: deedmon@emory.edu, Phone: 404-727-5972, Fax: 404-727-2738. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. animal models, is generally used for the screening of potential drug candidates. However, recent quantitative studies have shown differences in inhibitor specificities and properties b...

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Section: Catalytic Activitysupporting

confidence: 71%

Section: Purification Of Rmaobmentioning

confidence: 99%

Section: Membrane Localization and Purification Of Rmaobmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Characterization of detergent purified recombinant rat liver monoamine oxidase B expressed in Pichia pastoris

Upadhyay

Edmondson

2008

Protein Expression and Purification

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Structure and Mechanism of Monoamine Oxidase

Edmondson

Mattevi

Binda

et al. 2005

Burger's Medicinal Chemistry and Drug Discovery

113

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Monoamine oxidases A and B (MAO A and MAO B) are mitochondrial outer membrane‐bound flavoproteins that catalyze the oxidative deamination of neurotransmitters and biogenic amines. A number of mechanism‐based inhibitors (MAOIs) have been developed for clinical use as antidepressants and as neuroprotective drugs. To facilitate the development of more effective and specific inhibitors, a detailed understanding of the structures and catalytic mechanisms of these enzymes is required. The recent development of high level expression systems for producing recombinant human liver MAO A and MAO B in Pichia pastoris has facilitated the determination of the three‐dimensional crystal structures of MAO B (up to 1.7 Å resolution) in complex with different reversible (isatin, 1,4‐diphenyl‐2‐butene) and irreversible inhibitors (pargyline, N ‐(2‐aminoethyl)‐ p ‐chlorobenzamide, and trans ‐2‐phenylcyclopropylamine). The binding of substrates or inhibitors to MAO B involves an initial negotiation of a protein loop occurring near the surface of the membrane and two hydrophobic cavities; an “entrance” cavity and an “active site” cavity. These two cavities can either be separate or in a fused state depending on the conformation of the Ile199 side chain, which appears to function as a gate. The amine function of the bound substrate approaches the re face of the bent and “puckered” covalent FAD through an “aromatic cage” formed by two tyrosine residues that are perpendicular to the plane of the flavin ring. No amino acid residues that could function as acids or bases are found near the catalytic site. The existing structural data on MAO B support previous QSAR results and are also supportive of a proposed polar nucleophilic mechanism for MAO A and B catalysis rather than the alternatively proposed single electron transfer (SET) mechanism.

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EVBSimulations of the Catalytic Activity of Monoamine Oxidases: From Chemical Physics to Neurodegeneration

Vianello

Mavri

2017

Theory and Applications of the Empirical Valence Bond Approach

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High-Level Expression of Human Liver Monoamine Oxidase B in Pichia pastoris

Cited by 108 publications

References 46 publications

Characterization of detergent purified recombinant rat liver monoamine oxidase B expressed in Pichia pastoris

Characterization of detergent purified recombinant rat liver monoamine oxidase B expressed in Pichia pastoris

Structure and Mechanism of Monoamine Oxidase

EVBSimulations of the Catalytic Activity of Monoamine Oxidases: From Chemical Physics to Neurodegeneration

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