Abstract
Meprins are zinc-dependent proteases of the metzincin superfamily of metalloproteases. The enzymes are extracellular multi-domain proteins which are stabilized by disulfide bridges, dimerization, and glycosylation. Due to their complex structure, recombinant expression was first established in mammalian and insect cells. However, these methods have several disadvantages such as high costs and the low yields. For this reason, yeast is often considered a preferable expression system. Here, we describe the manipulation and secretory expression of human meprin β in the methylotrophic yeast P. pastoris. We show that the position of the affinity tag strongly influences the yield of expression, favoring fusion of the affinity tag at the C-terminus.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Dumermuth E, Sterchi EE, Jiang WP et al (1991) The astacin family of metalloendopeptidases. J Biol Chem 266(32):21381–21385
Sterchi EE, Stocker W, Bond JS (2008) Meprins, membrane-bound and secreted astacin metalloproteinases. Mol Aspects Med 29(5):309–328
Becker-Pauly C, Barre O, Schilling O et al (2011) Proteomic analyses reveal an acidic prime side specificity for the astacin metalloprotease family reflected by physiological substrates. Mol Cell Proteomics 10(9):M111.009233
Kronenberg D, Bruns BC, Moali C et al (2010) Processing of procollagen III by meprins: new players in extracellular matrix assembly? J Invest Dermatol 130(12):2727–2735
Bao J, Yura RE, Matters GL et al (2013) Meprin A impairs epithelial barrier function, enhances monocyte migration, and cleaves the tight junction protein occludin. Am J Physiol Renal Physiol 305(5):F714–F726
Huguenin M, Muller EJ, Trachsel-Rosmann S et al (2008) The metalloprotease meprinbeta processes E-cadherin and weakens intercellular adhesion. PLoS ONE 3(5):e2153
Broder C, Becker-Pauly C (2013) The metalloproteases meprin α and meprin β: unique enzymes in inflammation, neurodegeneration, cancer and fibrosis. Biochem J 450(2):253–264
Biasin V, Marsh LM, Egemnazarov B et al (2014) Meprin β, a novel mediator of vascular remodelling underlying pulmonary hypertension. J Pathol 233(1):7–17
Schlenzig D, Wermann M, Ramsbeck D et al (2015) Expression, purification and initial characterization of human meprin β from Pichia pastoris. Protein Expr Purif 116:75–81
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Schlenzig, D., Schilling, S. (2017). Heterologous Expression of the Astacin Protease Meprin β in Pichia pastoris . In: Galea, C. (eds) Matrix Metalloproteases. Methods in Molecular Biology, vol 1579. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6863-3_3
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6863-3_3
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6861-9
Online ISBN: 978-1-4939-6863-3
eBook Packages: Springer Protocols