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

Abstract

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...

Catalytic and inhibitor binding properties of zebrafish monoamine oxidase (zMAO): Comparisons with human MAO A and MAO B

“…In agreement with published data on hMAO A (Miller and Edmondson, 1999), on bovine MAO B (Walker and Edmondson, 1994), and on human and rat MAO B (Newton-Vinson, et al,2000;Li, 2006; Upadyhay and Edmondson, 2008), zMAO exhibits deuterium kinetic isotope effects on benzylamine analogue oxidation with D k cat and D ( k cat /K m ) values ranging from 2 to 8 (Table 4). These data demonstrate the α-C-H bond cleavage step contributes to the rate limitation in catalysis as is found with MAO A and B from various mammalian sources (Edmondson, 2009).…”

“…Previous work from this laboratory has shown the successful expression, purification, and partial characterization of recombinant zMAO (Arslan and Edmondson, 2009). This laboratory has also expressed, purified, and characterized human and rat MAO A and MAO B (Newton-Vinson, et al 2000; Li, et al 2002; Upadhyay and Edmondson 2008; Wang and Edmondson, 2009). Therefore, the tools are in place for a detailed comparative functional study of zMAO with those of human MAO A and MAO B.…”

Topological Probes of Monoamine Oxidases A and B in Rat Liver Mitochondria: Inhibition by TEMPO-Substituted Pargyline Analogues and Inactivation by Proteolysis

“…Recombinant rat liver MAO A and MAO B were expressed and purified in Pichia pastoris strain KM71 as described previously (17, 18). The purified enzymes were stored in 50 mM potassium phosphate containing 50% (w/v) glycerol and 0.8% (w/v) β–octylglucopyranoside (pH 7.2) at −20°C.…”

Expression of zebrafish (Danio rerio) monoamine oxidase (MAO) in Pichia pastoris: Purification and comparison with human MAO A and MAO B

“…Preliminary trials showed zMAO activity does not bind to DEAE Sepharose or to BioRad High-Q resin in contrast to what is observed with human or rat MAO A and MAO B [5-7]. Therefore, an alternate column purification procedure was adopted.…”

“…To aid in this effort, this laboratory has developed high level expression systems and purification protocols for the expression and purification of human and rat MAO A and of MAO B [5-7]. The rational for this work is to provide a bridge of knowledge between animal models (the rat) with the human in MAO inhibitor development.…”