Expression, purification and initial characterization of human meprin β from Pichia pastoris.

Abstract

Human meprin β (h-meprin β), a single-zinc metalloendoprotease of the astacin family, is potentially involved in disorders such as fibrosis and Alzheimer's disease. Here, we describe the expression of the enzyme in the yeast Pichia pastoris. The N-terminal signal sequence was replaced by the α-leader of Saccharomyces, enabling efficient secretion of the mature enzyme, harboring either an N-terminal or C-terminal His-tag. The purification by affinity and hydrophobic interaction chromatography resulted in isolation of 58.4 mg/l of homogenous human pro-meprin β from fermentation broth. The activated enzyme isolated from yeast (yh-meprin β) displayed virtually identical enzymatic activity as h-meprin from a mammalian cell line. Furthermore, the yh-meprin β was N-glycosylated and secreted as a dimer with a molecular mass of 148 kDa. Endoglycosidase H treatment generated a protein with a molecular mass of 133 kDa, but essentially unchanged kinetic parameters. Thus, our data suggest that human meprin β expressed in P. pastoris displays virtually identical parameters as meprin from other sources. The high yield of protein expression, the ease of purification and the deglycosylation in its native state appear to favor further studies aiming at inhibitor screening and structure-based inhibitor refinement.

• Authors: D Schlenzig, M Wermann, D Ramsbeck, T Moenke-Wedler, S Schilling
• Journal: Protein expression and purification (Protein Expr Purif) Vol. 116 Pg. 75-81 (Dec 2015) ISSN: 1096-0279 [Electronic] United States
• PMID: 26256061 (Publication Type: Journal Article)
• Copyright: Copyright © 2015 Elsevier Inc. All rights reserved.

Chemical References

• Recombinant Proteins
• Metalloendopeptidases
• meprin A

Topics

• Cloning, Molecular (methods)
• Electrophoresis, Polyacrylamide Gel
• Glycosylation
• Humans
• Metalloendopeptidases (chemistry, genetics, isolation & purification, metabolism)
• Pichia (genetics)
• Protein Multimerization
• Recombinant Proteins (chemistry, genetics, isolation & purification, metabolism)
• Substrate Specificity