title: Isolation of Metarhizium anisopliae carboxypeptidase A with native disulfide bonds from the cytosol of Escherichia coli BL21(DE3).
abstract: The carboxypeptidase A enzyme from Metarhizium anisopliae (MeCPA) has broader specificity than the mammalian A-type carboxypeptidases, making it a more useful reagent for the removal of short affinity tags and disordered residues from the C-termini of recombinant proteins. When secreted from baculovirus-infected insect cells, the yield of pure MeCPA was 0.25mg per liter of conditioned medium. Here, we describe a procedure for the production of MeCPA in the cytosol of Escherichia coli that yields approximately 0.5mg of pure enzyme per liter of cell culture. The bacterial system is much easier to scale up and far less expensive than the insect cell system. The expression strategy entails maintaining the proMeCPA zymogen in a soluble state by fusing it to the C-terminus of maltose-binding protein (MBP) while simultaneously overproducing the protein disulfide isomerase DsbC in the cytosol from a separate plasmid. Unexpectedly, we found that the yield of active and properly oxidized MeCPA was highest when coexpressed with DsbC in BL21(DE3) cells that do not also contain mutations in the trxB and gor genes. Moreover, the formation of active MeCPA was only partially dependent on the disulfide-isomerase activity of DsbC. Intriguingly, we observed that most of the active MeCPA was generated after cell lysis and amylose affinity purification of the MBP-proMeCPA fusion protein, during the time that the partially purified protein was held overnight at 4°C prior to activation with thermolysin. Following removal of the MBP-propeptide by thermolysin digestion, active MeCPA (with a C-terminal polyhistidine tag) was purified to homogeneity by immobilized metal affinity chromatography (IMAC), ion exchange chromatography and gel filtration.
authors.affiliation: Protein Engineering Section, 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: David S
authors.initials: DS
authors.lastname: Waugh
mesh_terms: D000595:Amino Acid Sequence; D016367:Baculoviridae / Q000235:genetics; D043422:Carboxypeptidases A / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D052981:Metarhizium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012995:Solubility
chemical_list: D004220:Disulfides; D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D043422:Carboxypeptidases A