Purification of recombinant endoglucanase GH5-CBM72-CBM72 expressed in Escherichia coli
Author affiliations
DOI:
https://doi.org/10.15625/2615-9023/16648Keywords:
Endoglucanase GH5CBM72CBM72, E. coli Rosetta 1, expression, purification, zymogramAbstract
In a previous study, a gene GL0694641 coding for endoglucanase containing 3 domains GH5-CBM72-CBM72 was exploited from metagenomic DNA data of bacteria in Vietnamese goats’ rumen. The gene (eg5) encoding the mature enzyme (without signal peptide coding sequence) was optimized codons, artificially synthesized, and inserted into the pET22b(+) vector at NcoI and XhoI to generate expression vector pET22-eg5 for expression of the gene in Eschrichia coli. In this study, the gene eg5 was well expressed in E. coli BL21 and Rosetta 1 strains to produce recombinant endoglucanase of 77 kDa. The recombinant enzymes were expressed mainly in the soluble fractions of both strains. However, the enzyme expressed in E. coli BL21 was precipitated by imidazole at even a low concentration of 20 mM, whereas endoglucanase produced from E. coli Rosetta 1 strain was well soluble in buffers containing imidazole at concentrations of 20, 50, 200, and 250 mM. To our knowledge, this is the first study showing the negative effect of imidazole on recombinant protein. Endoglucanase expressed from strain E. coli Rosetta 1 was successfully purified by His-tag affinity chromatography using phosphate buffer saline (PBS). Protein contaminations were washed out by PBS buffer containing 20 mM and 75 mM imidazole then the target protein was harvested by the buffer containing 200 mM imidazole. After purification and desalting by the PD10 column, the recombinant endoglucanase had purity up to 97%. The pure enzyme exhibited endoglucanase activity hydrolyzing carboxymethyl cellulose in agar plate and by zymogram. The purified enzyme can be used as a material for its characterization.
Downloads
Metrics
References
Amore A., Pepe O., Ventorino V., Birolo L., Giangrande C., Faraco V., 2012. Cloning and recombinant expression of a cellulase from the cellulolytic strain Streptomyces sp. G12 isolated from compost. Microb. Cell Factories, 11(1): 164. https://doi.org/ 10.1186/1475-2859-11-164
Bischoff K. M., Rooney A. P., Li X. L., Liu S., Hughes S.R., 2006. Purification and characterization of a family 5 endoglucanase from a moderately thermophilic strain of Bacillus licheniformis. Biotechnol. Lett., 28(21): 1761–1765. https://doi.org/10.1007/ s10529-006-9153-0
Boyce A., Walsh G., 2015. Characterisation of a novel thermostable endoglucanase from Alicyclobacillus vulcanalis of potential application in bioethanol production. Appl. Microbiol. Biotechnol., 99(18): 7515–7525. https://doi.org/ 10.1007/s00253-015-6474-8
Cano-Ramírez C., Santiago-Hernández A., Rivera-Orduña F. N., García-Huante Y., Zúñiga G., Hidalgo-Lara M. E., 2016. Expression, purification and characterization of an endoglucanase from Serratia proteamaculans CDBB-1961, isolated from the gut of Dendroctonus adjunctus (Coleoptera: Scolytinae). AMB Express, 6(1): 63. https://doi.org/ 10.1186/s13568-016-0233-9
Ding S. Y., Xu Q., Crowley M., Zeng Y., Nimlos M., Lamed R., Bayer E. A., Himmel M. E., 2008. A biophysical perspective on the cellulosome: new opportunities for biomass conversion. Curr. Opin. Biotechnol., 19(3): 218–227. https://doi.org/10.1016/j.copbio.2008.04.008
Do T. H., Dao T. K., Nguyen K. H. V., Le N. G., Nguyen T. M. P., Le T. L., Phung T. N., van Straalen N. M., Roelofs D., Truong N. H., 2018. Metagenomic analysis of bacterial community structure and diversity of lignocellulolytic bacteria in Vietnamese native goat rumen. Asian-Australas. J. Anim. Sci., 31(5): 738–747. https://doi.org/10.5713/ajas.17.0174
Du F., Liu Y. Q., Xu Y. S., Li Z. J., Wang Y. Z., Zhang Z. X., Sun X. M., 2021. Regulating the T7 RNA polymerase expression in E. coli BL21 (DE3) to provide more host options for recombinant protein production. Microb. Cell Factories, 20(1):189. https://doi.org/ 10.1186/s12934-021-01680-6
Duan C. J., Feng Y. L., Cao Q. L., Huang M. Y., Feng J. X., 2016. Identification of a novel family of carbohydrate-binding modules with broad ligand specificity. Sci. Rep., 6: 19392. https://doi.org/ 10.1038/srep19392
Guillén D., Sánchez S., Rodríguez-Sanoja R., 2010. Carbohydrate-binding domains: multiplicity of biological roles. Appl. Microbiol. Biotechnol., 85(5): 1241–1249. https://doi.org/10.1007/s00253-009-2331-y
Hamilton S., Odili J., Pacifico M. D., Wilson G. D., Kupsch J. M., 2003. Effect of imidazole on the solubility of a his-tagged antibody fragment. Hybrid. Hybridomics, 22(6): 347–355. https://doi.org/10.1089/ 153685903771797048
Holt S. M., Hartman P. A., 1994. A zymogram method to detect endoglucanases from Bacillus subtilis, Myrothecium verrucaria and Trichoderma reesei. J. Ind. Microbiol., 13(1): 2–4. https://doi.org/0.1007/BF01569654
Joshi N., Kaushal G., Singh S. P., 2021. Biochemical characterization of a novel thermo-halo-tolerant GH5 endoglucanase from a thermal spring metagenome. Biotechnol. Bioeng., 118(4): 1531–1544. https://doi.org/10.1002/bit.27668
Kang Y., Son M. S., Hoang T. T., 2007. One step engineering of T7-expression strains for protein production. Protein Expr. Purif., 55(2): 325–333. https://doi.org/ 0.1016/j.pep.2007.06.014
Kasana R. C., Salwan R., Dhar H., Dutt S., Gulati A., 2008. A rapid and easy method for the detection of microbial cellulases on agar plates using Gram’s iodine. Curr. Microbiol., 57(5): 503–507. https://doi.org/ 10.1007/s00284-008-9276-8
Lynd L.R., Weimer P. J., van Zyl W. H., Pretorius I. S., 2002. Microbial cellulose utilization: fundamentals and biotechnology. Microbiol. Mol. Biol. Rev. MMBR 66(3): 506–577. https://doi.org/ 10.1128/mmbr.66.3.506-577.2002
Nguyen H. D., Do T. H., Nguyen T. K., Ha T. T. H., Le Q. G., Dao T. K., Truong N. H., 2021. Expression of gene coding endoglucanase GH5-4 derived from metagenomic DNA data of bacteria in goats rumen in Escherichia coli. Acad. J. Biol., 43(2):17–26
Nguyen K. H. V., Dao T. K., Nguyen H. D., Nguyen K. H., Nguyen T. Q., Nguyen T. T., Nguyen T. M. P., Truong N. H., Do T. H., 2021. Some characters of bacterial cellulases in goats’ rumen elucidated by metagenomic DNA analysis and the role of fibronectin 3 module for endoglucanase function. Anim. Biosci., 34(5): 867–879. https://doi.org/10.5713/ajas.20.0115
Nguyen K. H. V., Nguyen T. T., Truong N. H., Do T. H., 2019. Application of bioinformatic tools for prediction of active pH and temperature stability of endoglucanases based on coding sequences from Metagenomic DNA data. Biological Forum, 11(2): 14–20.
Pandey S., Kushwah J., Tiwari R., Kumar R., Somvanshi V. S., Nain L., Saxena A. K., 2014. Cloning and expression of β-1, 4-endoglucanase gene from Bacillus subtilis isolated from soil long term irrigated with effluents of paper and pulp mill. Microbiol. Res., 169(9–10): 693–698. https://doi.org/10.1016/j.micres.2014.02.006
Rhodes D. G., Laue T. M., 2009. Chapter 38 Determination of protein purity. In Methods in Enzymology, Guide to Protein Purification, 2nd Edition, R. R. Burgess, M. P. Deutscher, eds, Academic Press, pp. 677–689.
Sharma P., Guptasarma P., 2017. Endoglucanase activity at a second site in Pyrococcus furiosus triosephosphate isomerase—Promiscuity or compensation for a metabolic handicap? FEBS Open Bio, 7(8): 1126–1143. https://doi.org/ 10.1002/2211-5463.12249
Shi R., Li Z., Ye Q., Xu J., Liu Y., 2013. Heterologous expression and characterization of a novel thermo-halotolerant endoglucanase Cel5H from Dictyoglomus thermophilum. Bioresour. Technol., 142: 338–344. https://doi.org/ 10.1016/j.biortech.2013.05.037
Vadala B. S., Deshpande S., Apte-Deshpande A., 2021. Soluble expression of recombinant active cellulase in E. coli using B. subtilis (natto strain) cellulase gene. J. Genet. Eng. Biotechnol., 19(1): 7. https://doi.org/10.1186/s43141-020-00103-0
Downloads
Published
How to Cite
Issue
Section
