Login Register Cart 0
Generic placeholder image

Current Biotechnology

Editor-in-Chief

ISSN (Print): 2211-5501
ISSN (Online): 2211-551X

Back
Journal Home About Journal Editorial Board Journal Insight Current Issue Volumes/Issues
Subscribe
Research Article

Preparation and Characterization of scFv-Coupled Immunoaffinity Column for Purification of Fibrinolytic Enzyme from Bacillus subtilis Natto-89

Author(s): SunIl Choe*, CholJin Kim, UnHui Yun, HyonGwang Li and CholHo Kim

Volume 9, Issue 2, 2020

Page: [104 - 110] Pages: 7

DOI: 10.2174/2211550109999200720162747

Price: $65

Abstract

Background: The focus of this study was to prepare and characterize the single-chain variable fragment antibody (scFv)-coupled immunoaffinity column for purification of subtilisin BRC.

Methods: The scFv against subtilisin BRC was immobilized onto CNBr-activated Sepharose 4B. Adsorption isotherm for subtilisin BRC on scFv-BRC-coupled Sepharose 4B was obtained and calculated the maximum binding capacity. The extraction conditions, including eluting solution, the concentration of eluting solution and flow rate, were optimized. Under the optimized eluting conditions, the dynamic binding capacity of the immunoaffinity column was determined.

Results: The scFv-BRC-coupled Sepharose 4B for immunoaffinity purification of subtilisin BRC was prepared. The coupling efficiency was about 78.4%, e.g. about 8 mg of scFv-BRC was covalently coupled to 1 g CNBr-activated Sepharose 4B. The maximum equilibrium binding capacity (qm) and dissociation constant (Kd) of the immunoaffinity column for subtilisin BRC were 3.01 mg/mL and 0.465 mg/mL, respectively. The immunoaffinity chromatography conditions were optimized and the subtilisin BRC was purified 3.29-fold with 55.6%.

Conclusion: The subtilisin BRC was effectively purified with high purity using scFv-BRC-coupled Sepharose 4B and the dynamic binding capacity of the column was determined. These results suggested that scFv-BRC can be used as a ligand for affinity purification of subtilisin BRC.

Keywords: Subtilisin BRC, fibrinolytic enzyme, scFv, immunoaffinity chromatography, dynamic binding capacity, Bacillus subtilis natto-89.

Next »
Graphical Abstract

[1]
Venkataraman D, Ilangovan S, Sampathkumar MV, et al. Medium optimization and immobilization of purified fibrinolytic URAK from Bacillus cereus NK1 on PHB nano-particles. Enzyme Microb Technol 2010; 47(6): 297-304.
[http://dx.doi.org/10.1016/j.enzmictec.2010.07.004]
[2]
Duffy MJ. Urokinase plasminogen activator and its inhibitor, PAI-1, as prognostic markers in breast cancer: From pilot to level 1 evidence studies. Clin Chem 2002; 48(8): 1194-7.
[http://dx.doi.org/10.1093/clinchem/48.8.1194] [PMID: 12142372]
[3]
Collen D, Lijnen HR. Tissue-type plasminogen activator: A historical perspective and personal account. J Thromb Haemost 2004; 2(4): 541-6.
[http://dx.doi.org/10.1111/j.1538-7933.2004.00645.x] [PMID: 15102005]
[4]
Wang XM, Fan SC, et al. Earthworm protease in anti-thrombosis and anti-fibrosis BBA – Gen Subjects 2019; 2(1863): 379-83.
[http://dx.doi.org/10.1016/j.bbagen.2018.11.006]
[5]
Sanchez EF, Richardson M, Gremski LH, et al. A novel fibrinolytic metalloproteinase, barnettlysin-I from Bothrops barnetti (barnett´s pitviper) snake venom with anti-platelet properties BBA – Gen Subjects 2016; 1860(3): 542-6.
[6]
Dametto M, David AP, Azzolini SS, et al. Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae). J Protein Chem 2000; 19(6): 515-21.
[http://dx.doi.org/10.1023/A:1026557600429] [PMID: 11195976]
[7]
Choi JH, Kim DW, Kim S, Kim SJ. Purification and partial characterization of a fibrinolytic enzyme from the fruiting body of the medicinal and edible mushroom Pleurotus ferulae. Prep Biochem Biotechnol 2017; 47(6): 539-46.
[http://dx.doi.org/10.1080/10826068.2016.1181083] [PMID: 27136080]
[8]
Kang SR, Choi JH, Kim DW, et al. A bifunctional protease from green alga Ulva pertusa with anticoagulant properties: Partial purification and characterization. J Appl Phycol 2016; 28(1): 599-607.
[http://dx.doi.org/10.1007/s10811-015-0550-4]
[9]
Kim JB, Jung WH, Ryu JM, et al. Identification of a fibrinolytic enzyme by Bacillus vallismortis and its potential as a bacteriolytic enzyme against Streptococcus mutans. Biotechnol Lett 2007; 29(4): 605-10.
[http://dx.doi.org/10.1007/s10529-006-9284-3] [PMID: 17308884]
[10]
Choi NS, Yoo KH, Hahm JH, et al. Purification and characterization of a new peptidase, Bacillopeptidase DJ-2, having fibrinolytic activity: Produced by Bacillus sp. DJ-2 from Doen-Jang. J Microbiol Biotechnol 2005; 15(1): 72-9.
[11]
Sumi H, Hamada H, Tsushima H, Mihara H, Muraki H. A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; a typical and popular soybean food in the Japanese diet. Experientia 1987; 43(10): 1110-1.
[http://dx.doi.org/10.1007/BF01956052] [PMID: 3478223]
[12]
Peng Y, Huang Q, Zhang RH, Zhang YZ. Purification and characterization of a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4 screened from douchi, a traditional Chinese soybean food. Comp Biochem Physiol B Biochem Mol Biol 2003; 134(1): 45-52.
[http://dx.doi.org/10.1016/S1096-4959(02)00183-5] [PMID: 12524032]
[13]
Achmad TP, Isnaeni P. Thrombolytic activity of fibrinolytic enzyme from black soybean tempeh (Glycine soja Sieb. Et Zucc) fermented by Rhizopus oligosporus FNCC 6010. Res J Pharm Biol Chem Sci 2017; 8(1): 1885-96.
[14]
Huy DNA, Hao PA, et al. Screening and identification of Bacillus sp. Isolated from traditional Vietnamese soybean-fermented products for high fibrinolytic enzyme production. Int Food Res J 2016; 23(1): 326-31.
[15]
Li HG, Li SG, Hyon C, Choe SI. Physiological and biochemical study on the multiple functions of “Chonggokkinase”. Proceeding of ISPHTI ‘08 ISMI ’08 Shenyang Forum 08. 2008 Oct 13-16; Shenyang, China. 2009.
[16]
Burgess RR, Thompson NE. Advances in gentle immunoaffinity chromatography. Curr Opin Biotechnol 2002; 13(4): 304-8.
[http://dx.doi.org/10.1016/S0958-1669(02)00340-3] [PMID: 12323350]
[17]
Alshammari TM, Al-Hassan AA, Hadda TB, Aljofan M. Comparison of different serum sample extraction methods and their suitability for mass spectrometry analysis. Saudi Pharm J 2015; 23(6): 689-97.
[http://dx.doi.org/10.1016/j.jsps.2015.01.023] [PMID: 26702265]
[18]
Blank K, Lindner P, Diefenbach B, Plückthun A. Self-immobilizing recombinant antibody fragments for immunoaffinity chromatography: Generic, parallel, and scalable protein purification. Protein Expr Purif 2002; 24(2): 313-22.
[http://dx.doi.org/10.1006/prep.2001.1575] [PMID: 11858727]
[19]
Guo Z, Bi F, Tang Y, et al. Preparation and characterization of scFv for affinity purification of reteplase. J Biochem Biophys Methods 2006; 67(1): 27-36.
[http://dx.doi.org/10.1016/j.jbbm.2005.12.007] [PMID: 16503053]
[20]
Geuijen CA, Bijl N, Smit RC, et al. A proteomic approach to tumour target identification using phage display, affinity purification and mass spectrometry. Eur J Cancer 2005; 41(1): 178-87.
[http://dx.doi.org/10.1016/j.ejca.2004.10.008] [PMID: 15618003]
[21]
Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: Principles and clinical application. Clin Dev Immunol 2012; 2012980250
[http://dx.doi.org/10.1155/2012/980250] [PMID: 22474489]
[22]
Kim CJ, Choe SI, Ri KC, et al. Selection of a single chain variable fragment antibody (scFv) against subtilisin BRC and its interaction with subtilisin BRC. Curr Biotechnol 2019; 8(1): 24-31.
[http://dx.doi.org/10.2174/2211550108666190417113342]
[23]
Kavran JM, Leahy DJ. Coupling antibody to cyanogen bromide-activated sepharose. Methods Enzymol 2014; 541: 27-34.
[http://dx.doi.org/10.1016/B978-0-12-420119-4.00003-3] [PMID: 24674060]
[24]
John MW. The Protein Protocols Handbook. 3rd ed. New York: Human Press 2009; pp. 17-24.
[25]
Boi C, Malavasi A, Carbonell RG, Gilleskie G. A direct comparison between membrane adsorber and packed column chromatography performance. J Chromatogr A 2020.1612460629
[http://dx.doi.org/10.1016/j.chroma.2019.460629] [PMID: 31668416]
[26]
Niu R, Yang Y, Wang S, et al. Chitosan microparticle-based immunoaffinity chromatography supports prepared by membrane emulsification technique: Characterization and application. Int J Biol Macromol 2019; 131: 1147-54.
[http://dx.doi.org/10.1016/j.ijbiomac.2019.04.064] [PMID: 30981776]
[27]
Agrebi R, Haddar A, Hajji M, et al. Fibrinolytic enzymes from a newly isolated marine bacterium Bacillus subtilis A26: Characterization and statistical media optimization. Can J Microbiol 2009; 55(9): 1049-61.
[http://dx.doi.org/10.1139/W09-057] [PMID: 19898547]
[28]
Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970; 227(5259): 680-5.
[http://dx.doi.org/10.1038/227680a0] [PMID: 5432063]
[29]
Chen JP, Wang CH. Microfiltration affinity purification of lactoferrin and immunoglobulin G from cheese whey. J Food Sci 1991; 56(3): 701-6.
[http://dx.doi.org/10.1111/j.1365-2621.1991.tb05360.x]
[30]
Firer MA. Efficient elution of functional proteins in affinity chromatography. J Biochem Biophys Methods 2001; 49(1-3): 433-2.
[http://dx.doi.org/10.1016/S0165-022X(01)00211-1] [PMID: 11694292]
[31]
Abdolalizadeh J, Majidi Zolbanin J, Nouri M, et al. Affinity purification of tumor necrosis factor-α expressed in raji cells by produced scFv Antibody coupled CNBr-Activated sepharose. Adv Pharm Bull 2013; 3(1): 19-23.
[PMID: 24312807]

Rights & Permissions Print Cite
Article Metrics
11
© 2024 Bentham Science Publishers | Privacy Policy