Carboxypeptidase G2 is a bacterial enzyme that catalyzes methotrexate conversion to its inactive forms which are then eliminated via a non-renal pathway in patients with renal disorders during a high-dose methotrexate administration. Due to the increasing demand of this enzyme, it was of interest to simplify its production process. For this reason, we developed a method for production and one-step purification of this enzyme using an intein-mediated system with a chitin-binding affinity tag. The carboxypeptidase G2 gene from Pseudomonas RS16 was optimized, synthesized, cloned into the pTXB1 expression vector and finally transformed into Escherichia coli BL21 (DE3) cells. The optimal condition for the enzyme soluble expression was achieved in 2×YT medium containing 1% glucose at 25°C for 30 h with 0.5 mM IPTG. The enzyme without intein was expressed as inclusion bodies indicating the importance of intein for the protein solubility. The expressed homodimer protein was purified to homogeneity on a chitin affinity column. The Km and kcat values of 6.5 µM and 4.57 s–1, respectively, were obtained for the purified enzyme. Gel filtration analysis indicated that the resulting recombinant protein was a dimer of 83 kDa. Fluorescence and circular dichroism spectroscopy confirmed the enzyme tertiary and secondary structures, respectively. The use of intein-mediated system provided the possibility of the one-step carboxypeptidase G2 purification, paving the way to the application of this enzyme in pharmaceutics.
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Rowsell, S., Pauptit, R. A., Tucker, A. D., Melton, R. G., Blow, D. M., and Brick, P. (1997) Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy, Structure, 5, 337-347, https://doi.org/10.1016/S0969-2126(97)00191-3.
Goda, S. K., Rashidi, F. A. B., Fakharo, A. A., and Al-Obaidli, A. (2009) Functional overexpression and purification of a codon optimized synthetic glucarpidase (Carboxypeptidase G2) in Escherichia coli, Protein J., 28, 435-442, https://doi.org/10.1016/J.ENZMICTEC.2016.08.001.
Ramsey, L. B., Balis, F. M., O’Brien, M. M., Schmiegelow, K., Pauley, J. L., et al. (2018) Consensus guideline for use of glucarpidase in patients with high-dose methotrexate induced acute kidney injury and delayed methotrexate clearance, Oncologist, 23, 52-61.
Rattu, M. A., Shah, N., Lee, J. M., Pham, A. Q., and Marzella, N. (2013) Glucarpidase (voraxaze), a carboxypeptidase enzyme for methotrexate toxicity, P T, 38, 732-744.
Wingfield, P. T. (2015) Overview of the purification of recombinant proteins, Curr. Protoc. Protein Sci., 80, 6.1.1-6.1.35, https://doi.org/10.1002/0471140864.ps0601s80.
Rosano, G. L., and Ceccarelli, E. A. (2014) Recombinant protein expression in Escherichia coli: advances and challenges, Front. Microbiol., 5, 172, https://doi.org/10.3389/fmicb.2014.00172.
Goh, H. C., Sobota, R. M., Ghadessy, F. J., and Nirantar, S. (2017) Going native: Complete removal of protein purification affinity tags by simple modification of existing tags and proteases, Protein Expr. Purif., 129, 18-24, https://doi.org/10.1016/J.PEP.2016.09.001.
Li, Y. (2011) Self-cleaving fusion tags for recombinant protein production, Biotechnol. Lett., 33, 869-881, https://doi.org/10.1007/s10529-011-0533-8.
Fan, Y., Miozzi, J. M., Stimple, S. D., Han, T. C., and Wood, D. W. (2018) Column-free purification methods for recombinant proteins using self-cleaving aggregating tags, Polymers (Basel), 10, 468, https://doi.org/10.3390/polym10050468.
Wu, W. Y., Mee, C., Califano, F., Banki, R., and Wood, D. W. (2006) Recombinant protein purification by self-cleaving aggregation tag, Nat. Protoc., 1, 2257-2262, https://doi.org/10.1038/nprot.2006.314.
Arnau, J., Lauritzen, C., Petersen, G. E., and Pedersen, J. (2006) Current strategies for the use of affinity tags and tag removal for the purification of recombinant proteins, Protein Expr. Purif., 48, 1-13, https://doi.org/10.1016/J.PEP.2005.12.002.
Belfort, M., Stoddard, B. L., Wood, D. W., and Derbyshire, V. (2006). Homing endonucleases and inteins, Springer Science & Business Media.
Banki, R., and Wood, D. W. (2005) Inteins and affinity resin substitutes for protein purification and scale up, Microb. Cell Fact., 4, 1-6, https://doi.org/10.1186/1475-2859-4-32.
Lahiry, A., Fan, Y., Stimple, S. D., Raith, M., and Wood, D. W. (2018) Inteins as tools for tagless and traceless protein purification, J. Chem. Technol. Biotechnol., 93, 1827-1835, https://doi.org/10.1002/jctb.5415.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680.
Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248-254.
Rashidi, F. B., AlQhatani, A. D., Bashraheel, S. S., Shaabani, S., Groves, M. R., et al. (2018) Isolation and molecular characterization of novel glucarpidases: enzymes to improve the antibody directed enzyme pro-drug therapy for cancer treatment, PLoS One, 13, e0196254, https://doi.org/10.1371/journal.pone.0196254.
AlQahtani, A. D, Al-Mansoori, L., Bashraheel, S. S., Rashidi, F. B., Al-Yafei, A., et al. (2019) Production of “biobetter” glucarpidase variants to improve drug detoxification and antibody directed enzyme prodrug therapy for cancer treatment, Eur. J. Pharm. >Sci., 127, 79-91, https://doi.org/10.1016/J.EJPS.2018.10.014.
Wang, L, Kang, J. H., Kim, K. H., and Leeb, E. K. (2009) Expression of intein-tagged fusion protein and its applications in downstream processing, J. Chem. Technol. Biotechnol., 85, 11-18, https://doi.org/10.1002/jctb.2277.
Fong, B. A., Wu, W. Y., and Wood, D. W. (2010) The potential role of self-cleaving purification tags in commercial-scale processes, Trends Biotechnol., 28, 272-279, https://doi.org/10.1016/j.tibtech.2010.02.003.
Yachnin, B. J., and Khare, S. D. (2017) Engineering carboxypeptidase G2 circular permutations for the design of an autoinhibited enzyme, Protein Eng. Des., 30, 321-331, https://doi.org/10.1093/protein/gzx005.
Alishah, K., Asad, S., Khajeh, K., and Akbari, N. (2016) Utilizing intein-mediated protein cleaving for purification of uricase, a multimeric enzyme, Enzyme Microb. Technol., 93-94, 92-98, https://doi.org/10.1016/J.Enzmictec.2016.08.001.
Wood, D. W., Derbyshire, V., Wu, W., Chartrain, M., Belfort, M., and Belfort, G. (2000) Optimized single-step affinity purification with a self-cleaving intein applied to human acidic fibroblast growth factor, Biotechnol. Prog., 16, 1055-1063, https://doi.org/10.1021/bp0000858.
Sharma, S. S., Chong, S., and Harcum, S. W. (2006) Intein-mediated protein purification of fusion proteins expressed under high-cell density conditions in E. coli, J. Biotechnol., 125, 48-56, https://doi.org/10.1016/j.jbiotec.2006.01.018.
Díaz, M., Venturini, E., Marchetti, S., Arenas, G., and Marshall, S. H. (2012) Intein-mediated expression of cecropin in Escherichia coli, Electron. J. Biotechnol., 15, 1-10.
Sherwood, R. F, Melton, R. G., Alwan, S. M., and Hughes, P. (1985) Purification and properties of carboxypeptidase G2 from Pseudomonas sp. strain RS-16, Eur. J. Biochem., 148, 447-453, https://doi.org/10.1111/j.1432-1033.1985.tb08860.x.
Minton, N. P., Atkinson, T., and Sherwood, R. F. (1983) Molecular cloning of the Pseudomonas carboxypeptidase G2 gene and its expression in Escherichia coli and Pseudomonas putida, J. Bacteriol., 156, 1222-1227.
Jeyaharan, D., Aston, P., Garcia-Perez, A., Schouten, J., Davis, P., and Dixon, A. M. (2016) Soluble expression, purification and functional characterisation of carboxypeptidase G2 and its individual domains, Protein Expr. Purif., 127, 44-52, https://doi.org/10.1016/J.PEP.2016.06.015.
Agostini, F., Cirillo, D., Livi, C. M., DelliPonti, R., and Tartaglia, G. G. (2014) ccSOL omics: a webserver for solubility prediction of endogenous and heterologous expression in Escherichia coli, Bioinformatics, 30, 2975-2977, https://doi.org/10.1093/bioinformatics/btu420.
Hebditch, M., Carballo-Amador, M. A., Charonis, S., Curtis, R., and Warwicker, J. (2017) Protein-Sol: a web tool for predicting protein solubility from sequence, Bioinformatics, 33, 3098-3100, https://doi.org/10.1093/bioinformatics/btx345.
We gratefully appreciate the Research Council of Tarbiat Modares University, Prof. Khosro Khajeh, and Iran National Institute for Medical Research Development (NIMAD, project 940711) for their financial support through this investigation.
The authors declare no conflict of interest. This article does not contain description of studies with the involvement of humans or animal subjects.
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Khodakarami, A., Dabirmanesh, B., Asad, S. et al. Enhanced Solubility and One-Step Purification of Functional Dimeric Carboxypeptidase G2. Biochemistry Moscow 86, 190–196 (2021). https://doi.org/10.1134/S0006297921020073
- carboxypeptidase G2