Isolation of Metarhizium anisopliae carboxypeptidase A with native disulfide bonds from the cytosol of Escherichia coli BL21(DE3).

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.

High enrichment of MMP-9 and carboxypeptidase A by tweezing adsorptive bubble separation (TABS).

Tweezing adsorptive bubble separation (TABS) was used as a method for the enrichment of matrix metalloproteinases (92-kDa type IV, gelatinase B (MMP-9)) and carboxypeptidase A (CPA) from dilute aqueous solutions. The method is based on the chelation of metalloenzymes applying 2-(carbamoylmethyl-(carboxymethyl)amino)acetic acid (ADA) coupled with an octyl part to form a surface active unit. MMP-9 could be enriched with an enrichment ratio of 12.0 and a recovery of 87.3%, and CPA could be enriched 18.8-fold and with 95.3% recovery. Both enzymes were enriched without significant losses of enzymatic activity. To verify that the enzymes were tweezed by ADA-C8 without abstraction of the zinc ions from the active center, TABS trials were additionally conducted with zinc ions in complex with ADA-C8, which revealed only negligible enrichment ratios of the enzymes (2.2 for MMP-9 and 0.2 for CPA). The results obtained impressively demonstrate that zinc-containing proteases can be enriched selectively and efficiently by TABS.

Identification and distribution of mouse carboxypeptidase A-6.

Carboxypeptidase A-6 (CPA6) was recently discovered in the human genome. To gain information regarding the potential function of this novel protein, the mouse homolog of CPA6 was identified using a combination of bioinformatics and reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, homologs in rat, chicken, and frog were identified using a bioinformatics approach. The distribution of CPA6 mRNA in mouse tissues was examined using RT-PCR and in situ hybridization. A strong RT-PCR signal is detectable in olfactory bulb, and much lower levels are present in other regions such as the cerebral cortex, hippocampus, hypothalamus, striatum, and medulla. In peripheral tissues, a moderate RT-PCR signal is present in epididymis, and low levels are detectable in colon and spleen. The high level of CPA6 in adult mouse brain olfactory bulb was confirmed by in situ hybridization. Lower levels of CPA6 mRNA were found to be present in the cingulate cortex, lateral septum, pontine nucleus, and inferior olivary nucleus of the hindbrain. Within the olfactory bulb, CPA6 mRNA is enriched in the mitral and granular layer. A lower level of CPA6 mRNA is present in the internal and external plexiform layers, and no signal is detectable in the olfactory nerve layer. The distribution was also examined in whole embryos at embryonic day 14.5 and CPA6 mRNA was found to be enriched in eye, ear, osteoblasts, stomach, skin, dorsal root ganglia, and throughout the CNS. The presence of CPA6 mRNA in the rectus muscle layer of the eye at embryonic day 14.5 is consistent with the observation that the CPA6 gene is disrupted in a patient with Duane syndrome, a congenital eye defect. Taken together, the distribution of CPA6 suggests a specific role in a limited number of tissues, and it is possible that this role involves an aspect of cell migration.