Comparison of Two Purification Methods for Beta-Toxin of Clostridium perfringens Type B

Document Type : Research Article

Authors

1 Department of Chemistry, Mashhad Branch, Islamic Azad University, Mashhad, I.R. IRAN

2 Education and Extension Organization (AREEO), Agricultural Research, Razi Vaccine and Serum Research Institute, Mashhad Branch, Mashhad, I.R. IRAN

Abstract

Clostridium perfringens is an anaerobic bacterium that among its strains, only types B and C can produce beta-toxin. Beta-toxin plays an important role in many human and animal diseases and has detrimental effects on the intestine. The present study aimed to compare two purification methods for the extraction of beta-toxin from C. perfringens type B. As the first method, beta-toxin was purified using ammonium sulfate precipitation, column chromatography (Sephadex G-25), and ion-exchange chromatography (DEAE Sephadex), while the second method employed affinity chromatography. Hemolysis activity and protein content were measured in each step of purification. The purity of beta-toxin was monitored in each step using SDS-PAGE. According to the results, a comparison of these two methods indicated that the yield of the first method was 82.8%, and the yield of the second method was 90.1%. The specific activity values for the first and second methods were calculated to be 2368.1 U/mg and 164.5 U/mg, respectively. Our results show that affinity chromatography could be used to purify beta-toxin from clostridium perfringens type B with high purity and specific activity (4370 HU/mg).

Keywords

Main Subjects

References

[1] Juneja VK., Marks H., Thippareddi HH., Predictive Model For Growth of Clostridium Perfringens During Cooling of Cooked Ground Pork, Innovative Food Sci. Emerging Technol., 11(1):146-154 (2010).
[2] Milach A., de los Santos J.R.G., Turnes C.G., Moreira Â.N., de Assis R.A., Salvarani F.M., et al., Production and Characterization of Clostridium Perfringens Recombinant $β$ Toxoid, Anaerobe, 18(3): 363-365 (2012).
[3] Sakurai J., Nagahama M., Clostridium Perfringens Beta-Toxin: Characterization and Action, Toxin Rev., 25(1):89-108 (2006).
[4] Bakhshi F., Langroudi R.P., Eimani B.G., Enhanced Expression of Recombinant Beta Toxin of Clostridium Perfringens Type B Using a Commercially Available Escherichia Coli Strain, Onderstepoort J. Vet. Res., 83(1):1-4 (2016).
[5] Navarro M.A., McClane B.A., Uzal F.A.J.T., Mechanisms of Action and Cell Death Associated With Clostridium Perfringens Toxins, Toxins., 10(5): 212 (2018).
[6] Zayerzadeh E., Fardipour A., Jabbari A., A New Purification Method for Beta-Toxin of Clostridium perfringens Type C Vaccinal Strain, J. Med. Bacteriol, 3(4):8-13 (2014).
[7] Cavalcanti M.T.H., Porto T., Porto A.L.F., Brandi I.V., Lima Filho J.L.d., Large Scale Purification of Clostridium Perfringens Toxins: A Review, Braz. J. Pharm. Sci., 40(2):151-164 (2004).
[8] Xiong N., Yu R., Chen T., Xue Y-P., Liu Z-Q., Zheng Y-G., Separation and Purification of L-Methionine from E. Coli Fermentation Broth by Macroporous Resin Chromatography, J. Chromatogr. B: Biomed. Sci. Appl., 1110:108-115 (2019).
[9] Coskun O., Separation Techniques: Chromatography, North Clin Istanb, 3(2):156 (2016).
[10] Gagnon P., Technology Trends in Antibody Purification, J. Chromatogr. A., 1221: 57-70 (2012).
[11] Zhao X., Li G., Liang S., Several Affinity Tags Commonly Used in Chromatographic Purification, J. Anal. Methods Chem., 2013 (2013).
[12] Fekete S., Beck A., Veuthey J-L., Guillarme D., Ion-Exchange Chromatography for the Characterization of Biopharmaceuticals, J. Pharm. Biomed. Anal., 113(1):43-55 (2015)
[13] Schmidt M., Hafner M., Frech C., Modeling of Salt and pH Gradient Elution in Ion-Exchange Chromatography, J. Sep. Sci., 37(2): 5-13 (2014).
[14] Bakhshi F., Langroudi RP., Eimani BG., Enhanced Expression of Recombinant Beta Toxin of Clostridium Perfringens Type B Using a Commercially Available Escherichia Coli Strain, Onderstepoort J. Vet. Res., 83(1):1-4 (2016).
[15] Sayadmanesh A., Ebrahimi F., Hajizade A., Rostamian M., Keshavarz H.J.I.B.J., Expression and Purification of Neurotoxin-Associated Protein HA-33/A from Clostridium Botulinum and Evaluation of Its Antigenicity, Iran. Biomed. J., 17(4):165 (2013).
[16]  Zayerzadeh E., Fardipour A., Jabbari AJJoMB., A New Purification Method for Beta-Toxin of Clostridium perfringens Type C Vaccinal Strain, J. Med. Bacteriol., 3(3-4):8-13 (2014).
[17] zahra heshmati Z., Fathi Najafi M., Ronaghi GH H., Purification and Characterization of Alpha Toxin Clostridium perfringens Type A, Physiology and Molecular Biology., 12(2):79-86 (2020).
[18] Johnson E., Tepp W., Lin G., Purification, Characterization, and use of Clostridium Botulinum Neurotoxin BoNT/A3, Google Patents; US9498521B2 (2016).
[19] Möllby R., Holme T., Nord C-E., Smyth C., Wadström TJM., Production of Pphospholipase C (alpha-toxin), Haemolysins and Lethal Toxins by Clostridium Perfringens Types A to D, Microbiology., 96(1):137-144 (1976).
[20] Bradford MMJAb, A Rapid And Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding, Anal. Biochem., 72(1-2): 248-254 (1976).
[21] Coskun O., Separation techniques: Chromatography, Northern Clinics of Istanbul, 3(2):156 (2016).
[22] Liao X., Zhang B., Wang X., Yan H., Zhang XJC., Purification of C-Phycocyanin from Spirulina Platensis by Single-Step Ion-Exchange Chromatography, Chromatographia, 73(3): 291-296 (2011).
[23] Selzer J., Hofmann F., Rex G., Wilm M., Mann M., Just I., et al., Clostridium Novyi α-toxin-catalyzed Incorporation of GlcNAc into Rho Subfamily Proteins, J. Biol. Chem., 271(41): 25173-25177 (1996).
[24] Gkogka E., Reij M.W., Gorris L.G., Zwietering M., Risk Assessment of Clostridium Perfringens in Cornish Pasties in the UK, Food Control., 108(1): 106822 (2020).
[25]. Mehdizadeh Gohari I., Li J., McClane B., Identifying the Basis for VirS/VirR Two-Component Regulatory System Control of Clostridium perfringens Beta-Toxin Production, J. Bacteriol., 203(18): 00279-00221 (2021).
[26] Nijland R., Lindner C., Van Hartskamp M., Hamoen L.W., Heterologous Production and Secretion of Clostridium Perfringens β-toxoid in Closely Related Gram-Positive Hosts, Acad. J. Biotechnol., 127(3): 361-372 (2007).
[27] Bhatia B., Solanki AK., Kaushik H., Dixit A., Garg L.C.J.Pe., B-Cell Epitope of Beta Toxin of Clostridium Perfringens Genetically Conjugated to a Carrier Protein: Expression, Purification and Characterization of the Chimeric Protein, Protein Expression Purif., 102: 38-44 (2014).
[28] Kouguchi H., Watanabe T., Sagane Y., Ohyama T., Characterization and Reconstitution Of Functional Hemagglutinin of the Clostridium Botulinum Type C Progenitor Toxin, Eur. J. Biochem., 268(14):4019-4026 (2001).
[29] Cavalcanti M.T.H., Porto T., Porto A.L.F., Brandi I.V., Lima Filho J.Ld., Large Scale Purification of Clostridium Perfringens Toxins: A Review, Rev. Bras. Cienc. Farm., 40:151-164 (2004).
Iranian Journal of Chemistry and Chemical Engineering
Volume 41, Issue 9 - Serial Number 119
September 2022
Pages 2911-2917

Files

Share

How to cite

Statistics