N-Succinyl-L,L-Diaminopimelic Acid Desuccinylase (DapE) a Potential Biomarker of Mycobacterium Tuberculosis: Tag-Free Purification by Metal Affinity Chromatography Tag-free purification of DapE by metal affinity chromatography
Trends in Peptide and Protein Sciences,
Vol. 5 (2020),
1 January 2020
,
Page 1-9 (e4)
https://doi.org/10.22037/tpps.v5i0.31578
Abstract
DapE is an enzyme which is highly essential in lysine biosynthetic pathway for the growth and survival of Mycobacterium tuberculosis (Mtb) and other bacterial species although absent in human host. However, the sequence identity of Mtb-DapE with other mycobacterium and bacterial species estimated between 93 to 75% and approximately 50%, respectively. DapE is a metallo-enzyme requires few of transition metals for activity and bacterial proliferation. The homology model based structural studies including published structures revealed an availability of zinc interacting conserved amino residues in Mtb-DapE. In this study, we purified full length recombinant Mtb-DapE as tag-free (Mtb-DapETagFree) protein from inclusion bodies using zinc-NTA column. The single step purified protein observed with 96-98% purity and high yield. The indirect ELISA had 40% sensitivity using Mtb-DapE as an antigen against bovine tuberculosis (bTB) serum samples. IFA analysis with clear fluorescent spots of Mtb native DapE antigen in (3+) human TB positive sputum samples and recombinant Mtb-DapE positive control against Mtb-DapE polyclonal antibody are highly encouraging. The ELISA and IFA results incite for the consideration of Mtb-DapE in future development of quick and ideal TB detection assay along with other mycobacterium antigens. The future advanced detailed structural and functional studies using this highly purified and tag-free Mtb-DapE may provide discovery of antitubercular drug(s) and promising inhibitory molecules.
HIGHLIGHTS
- Expression and purification of recombinant Mtb-DapETagFree.
- On-column refolded protein with 98% purity from inclusion bodies purified by zinc-NTA column affinity chromatography.
- The purified protein observed no aggregation and degradation even after 8 months storage.
- ELISA and IFA results supports for development of detection assay for TB from tubercular samples.
- Affinity chromatography
- DapE
- Mycobacterium tuberculosis
- Protein
How to Cite
References
Anindita, M., Thamke, D., Mendiratta, D. K. and B. C. Harinath, (2010). "Potential of Mycobacterial excretory secretory protein antigen (SEVA TB ES-31, ES-43, EST-6, ES-20) as a biomarker to detect Mycobacterium tuberculosis bacilli." Indian Journal of Clinical Biochemistry, 25: 15-19.
Bhusan, K. K., Polamarasetty, A., and R. Pallu, (2010). "EasyModeller: A graphical interface to MODELLER." BMC Research Notes, 3: 226.
Boguslaw, N. B., Reidl, C., Starus, A., Heath, T.,Bienvenue, D., Osipiuk, J., Jedrzejczak, R., Joachimiak, A., Becker, D.P. and R.C. Holz, (2018). "Structural Evidence of a Major Conformational Change Triggered by Substrate Binding in DapE Enzymes: Impact on the Catalytic Mechanism." Biochemistry, 57 (5): 574–584
Bowie, J.U., Lüthy, R. and D. A. Eisenberg, (1991). "Method to Identify Protein Sequences That Fold Into a Known Three-Dimensional Structure." Science, 12: 164-170.
Ceroni, A., Passerini, A., Vullo, A. and P. Frasconi, (2006). "DISULFIND: a Disulfide Bonding State and Cysteine Connectivity Prediction Server." Nucleic Acids Research, 34 (Web Server issue):177-181.
Chant A., Kraemer-Pecore C.M., Watkin R. and G. G. Kneale, (2005). "Attachment of a histidine tag to the minimal zinc finger protein of the Aspergillus nidulans gene regulatory protein AreA causes a conformational change at the DNA-binding site."Protein Expression and Purification, 39(2): 152-159.
Cimino, M., Lorenzo, A. L. and L. Salazar, (2006). "Permeabilization of the mycobacterial envelope for protein cytolocalization studies by immunofluorescence microscopy." BMC Microbiology, 6(35): 1-4.
Cooper, H. M. and Y. Paterson, (2000). "Determination of thw specific antibody titer." Current protocols in Molecular Biology, 50(1): 11.17.1-11.17.13.
DeLano, W. L. (2002). "Pymol: An open-source molecular graphics tool." CCP4 Newsletter On Protein Crystallography, 40: 82-92.
Fonda I., Kenig, M., Gaberc-Porekar, V., Pristovaek, P. and V. Menart, (2002). "Attachment of histidine tags to recombinant tumor necrosis factor-alpha drastically changes its properties." Scientific World Journal, 15(2): 1312–1325.
Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S., Wilkins, M.R., Appel, R.D. and A. Bairoch, (2005). "Protein Identification and Analysis Tools on the ExPASy Server." In: John M. Walker (Ed.), The Proteomics Protocols Handbook, Humana Press, pp. 571-607.
Geourjon, C. and G. Deleage, (1995). "SOPMA: Significant improvement in protein secondary structure prediction by c prediction from alignments and joint prediction." CABIOS, 11: 681-684.
Gillner, D., Armoush, N., Holz, R. C. and D. P. Becker, (2009). "Inhibitors of bacterial N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) and demonstration of in vitro antimicrobial activity."Bioorganic and Medicinal Chemistry Letters, 19(22): 6350–6352.
Greenfield, E. A. and E. Harlow, (2014). "Antibodies: a laboratory manual." In: E.A. Greenfield (Ed.), Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Leenaars, M. and C. F. M. Hendriksen, (2005). "Critical Steps in the Production of Polyclonal and Monoclonal Antibodies: Evaluation and Recommendations." ILAR Journal, 46 (3): 269-279.
Lüthy, R., Bowie, J.U. and D. Eisenberg, (1992). "Assessment of Protein Models With Three-Dimensional Profiles." Nature, 356(6364): 83-85.
Maxmen, A. (2019). "Treatment for extreme drug-resistant tuberculosis wins US government approval." Nature. Available at: https://www.nature.com/articles/d41586-019-02464-0
Nocek, B. P., Gillner, D. M., Fan, Y., Holz, R.C. and A. Joachimiak, (2010). "Structural basis for catalysis by the mono- and dimetalated forms of the dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase." Journal Molecular Biology, 397(3): 617-626.
Pettersen, E. F., Goddard, T.D., Huang, C.C., Couch, G. S., Greenblatt, D.M., Meng, E. C. and T. E. Ferrin, (2004). "UCSF Chimera--a visualization system for exploratory research and analysis." Journal of Computational Chemistry, 25(13): 1605-1612.
Reinhard, L., Mueller-Dieckmann, J. and M. Weiss, (2012). "Cloning, Expression, Purification, Crystallization and Preliminary X-ray Diffraction Analysis of Succinyl-Diaminopimelate Desuccinylase (Rv1202, DapE) From Mycobacterium Tuberculosis." Acta Crystallographica, F68: 1089–1093.
Singh, S.V., Stephen, B.J., Singh, M., Gupta, S., Chaubey, K.K., Jayaraman, S., Sachan, T.K., Aseri, G.K., Dutta, M., Sohal, J.S., Dhama, K., Mukartal, S.Y. and R. Doddamane, (2016). "Evaluation of indirect fluorescent antibody test as a potential screening test for Mycobacterium avium subspecies paratuberculosis using milk of lactating domestic livestock." Journal of Experimental Biology and Agricultural Sciences, 5: 533-540.
Starus, A., Boguslaw, N.B., Bennett, B., Larrabee, J.A., Shaw, D.L., Sae-Lee, W., Russo, M.T., Gillner, D.M., Makowska-Grzyska, M., Joachimiak, A., and R. C. Holz, (2015). "Inhibition of the dapE-Encoded N-Succinyl-L,L-diaminopimelic Acid Desuccinylase from Neisseria meningitidis by L-Captopril." Biochemistry, 54(31): 4834-4844.
Starus, A., Nocek, B., Bennett, B., Larrabee, J. A., Shaw, D. L., Lee, W.S., Russo, M. T., Gillner, D.M., Grzyska, M.M. , Joachimiak, A. and R. C. Holz, (2015). "Inhibition of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Neisseria meningitidis by L-captopril." Biochemistry, 54(31): 4834–4844.
Studer, G., Rempfer, C., Waterhouse, A.M., Gumienny, G., Haas, J., and T. Schwede, (2020). "QMEANDisCo - distance constraints applied on model quality estimation." Bioinformatics, 36: 1765-1771.
Towbin, H., Staehelin, T., and J. Gordon, (1979). "Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications." Proceedings of the National Academy of Sciences USA, 76(9): 4350–4354.
Uda, N. and M. Creus, (2011). "Selectivity of inhibition of N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) in bacteria:DapE is not the target of L-captopril antimicrobial activity. "Bioinorganic Chemistry and Applications, 2011(306465): 6 Pages.
Umbarkar, C. P., Thakre, P. S., Kashikar, S.A., Ghanwate, N. A. and V. B. Agarkar, (2019). "Cloning, expression, and refolding of N-succinyl-l-l diaminopimelic acid desuccinylase (MTB-DapE) of Mycobacterium tuberculosis." International Journal of Life Sciences Research, 7(3): 110-115.
Usha, V., Lloyd, A.J., Roper, D.I., Dowson, C.G., Kozlov G., Gehring, K., Chauhan, S., Imam, H. T., Blindauer, C.A. and G.S. Besra, (2016). "Reconstruction of diaminopimelic acid biosynthesis allows characterisation of Mycobacterium tuberculosis N-succinyl-L,L-diaminopimelic acid desuccinylase." Scientific Reports, 6 (23191): 1-10
Vasantha, M. and P.Venkatesan, (2014). "Structural equation modeling of latent growth curves of weight gain among treated tuberculosis patients." PLoS One, 9(3): e91152.
Wiederstein and Sippl, (2007). "ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins." Nucleic Acids Research, 35: W407-W410
- Abstract Viewed: 373 times
- PDF Downloaded: 195 times