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Protein Expr Purif. 2011 May;77(1):53-61. doi: 10.1016/j.pep.2010.11.005. Epub 2010 Nov 10.

The substrate specificity of Metarhizium anisopliae and Bos taurus carboxypeptidases A: insights into their use as tools for the removal of affinity tags.

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Macromolecular Crystallography Laboratory, Center for Cancer Research, National Cancer Institute at Frederick, P.O. Box B, Frederick, MD 21702-1201, USA.


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.

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