title: The substrate specificity of Metarhizium anisopliae and Bos taurus carboxypeptidases A: insights into their use as tools for the removal of affinity tags.
abstract: Carboxypeptidases may serve as tools for removal of C-terminal affinity tags. In the present study, we describe the expression and purification of an A-type carboxypeptidase from the fungal pathogen Metarhizium anisopliae (MeCPA) that has been genetically engineered to facilitate the removal of polyhistidine tags from the C-termini of recombinant proteins. A complete, systematic analysis of the specificity of MeCPA in comparison with that of bovine carboxypeptidase A (BoCPA) was carried out. Our results indicate that the specificity of the two enzymes is similar but not identical. Histidine residues are removed more efficiently by MeCPA. The very inefficient digestion of peptides with C-terminal lysine or arginine residues, along with the complete inability of the enzyme to remove a C-terminal proline, suggests a strategy for designing C-terminal affinity tags that can be trimmed by MeCPA (or BoCPA) to produce a digestion product with a homogeneous endpoint.
authors.affiliation: Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, P.O. Box B, Frederick, MD 21702-1201, USA.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Austin
authors.forename: József
authors.initials: J
authors.lastname: Tözsér
authors.forename: Péter
authors.initials: P
authors.lastname: Bagossi
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Tropea
authors.forename: David S
authors.initials: DS
authors.lastname: Waugh
mesh_terms: D000345:Affinity Labels / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D043422:Carboxypeptidases A / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle / Q000378:metabolism*; D006639:Histidine / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D052981:Metarhizium / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D012965:Sodium Chloride; D013379:Substrate Specificity
chemical_list: D000345:Affinity Labels; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D012965:Sodium Chloride; D006639:Histidine; D043422:Carboxypeptidases A