title: Soluble expression, purification and functional characterisation of carboxypeptidase G2 and its individual domains.
abstract: Due to its applications in the treatment of cancer and autoimmune diseases, the 42 kDa zinc-dependent metalloenzyme carboxypeptidase G2 (CPG2) is of great therapeutic interest. An X-ray crystal structure of unliganded CPG2 reported in 1997 revealed the domain architecture and informed early rational drug design efforts, however further efforts at co-crystallization of CPG2 with ligands, substrates or inhibitors have not been reported. Thus key features of CPG2 such as the location of the active site, the presence of additional ligand-binding sites, stability, oligomeric state, and the molecular basis of activity remain largely unknown, with the current working understanding of CPG2 activity based primarily on computational modelling. To facilitate renewed efforts in CPG2 structural biology, we report the first high-yield (250 mg L(-1)) recombinant expression (and purification) of soluble and active CPG2 using the Escherichia coli expression system. We used this protocol to produce full-length enzyme, as well as protein fragments corresponding to the individual catalytic and dimerization domains, and the activity and stability of each construct was characterised. We adapted our protocol to allow for uniform incorporation of NMR labels ((13)C, (15)N and (2)H) and present preliminary solution-state NMR spectra of high quality. Taken together, our results offer a route for production and solution-state characterization that supports renewed effort in CPG2 structural biology as well as design of significantly truncated CPG2 proteins, which retain activity while yielding (potentially) improved immunogenicity.
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK.
authors.forename: Dhadchayini
authors.initials: D
authors.lastname: Jeyaharan
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK.
authors.forename: Philip
authors.initials: P
authors.lastname: Aston
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK.
authors.forename: Angela
authors.initials: A
authors.lastname: Garcia-Perez
authors.affiliation: Mologic Ltd, Bedford Technology Park, Thurleigh, Bedford, MK44 2YP, UK.
authors.forename: James
authors.initials: J
authors.lastname: Schouten
authors.affiliation: Mologic Ltd, Bedford Technology Park, Thurleigh, Bedford, MK44 2YP, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Davis
authors.affiliation: Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK. Electronic address: ann.dixon@warwick.ac.uk.
authors.forename: Ann M
authors.initials: AM
authors.lastname: Dixon
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D019906:Nuclear Magnetic Resonance, Biomolecular; D000072417:Protein Domains; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011623:gamma-Glutamyl Hydrolase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D011623:gamma-Glutamyl Hydrolase
keywords: Active enzyme fragment; Activity measurements; Bacterial expression; Carboxypeptidase G2; Isotopic labelling; Mass spectrometry; Nuclear magnetic resonance