Volume 11, Issue 2 (Vol.11 No.2 Jul 2022)                   rbmb.net 2022, 11(2): 252-261 | Back to browse issues page

XML Print

Download citation:
BibTeX | RIS | EndNote | Medlars | ProCite | Reference Manager | RefWorks
Send citation to:

Danaeifar M, Veisi Malekshahi Z, Kazemi-Lomedasht F, Mazlomi M A. Recombinant Protein Purification using Composite Polyacrylamide-Nanocrystalline Cryogel Monolith Column and Carbohydrate- Binding Module Family 64 as Affinity Tag. rbmb.net 2022; 11 (2) :252-261
URL: http://rbmb.net/article-1-847-en.html
Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Abstract:   (1149 Views)
Background: In the field of recombinant protein production, downstream processing, especially protein purification, is critical and often the most expensive step. Carbohydrate binding module 64 (CBM64) was shown in 2011 to bind efficiently to a broad range of cellulose materials.

Methods: In this study, we developed a protein purification method using nanocrystalline cellulose embedded in a polyacrylamide monolith cryogel and CBM64 affinity tag linked by intein to PD1 as a model protein. The CBM64-Intein-PD1 gene cassette was expressed in E. coli. Following cell lysis, CBM64-Intein-PD1 protein bound to the monolith PA-NCC cryogel. After washing and reducing the pH from 8.0 to 6.5, the intein underwent self-cleavage, resulting in the release and elution of pure PD1 protein.

Results: The synthesized monolith column had a porous structure with an average pore size of 30 μm and a maximum binding capacity of 497 μg per gram of dried column. The yield of this purification method was 84%, while the yield of the His tag-acquired CBM64-Intein-PD1 method was 89%.

Conclusions: We used cellulose as support for affinity chromatography, which can be used as a cost-effective method for protein purification.
Full-Text [PDF 287 kb]   (444 Downloads)    
Type of Article: Original Article | Subject: Biochemistry
Received: 2021/12/28 | Accepted: 2021/12/29 | Published: 2022/08/7

1. Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front microbiol. 2014;5:172. [DOI:10.3389/fmicb.2014.00172]
2. Rege K, Pepsin M, Falcon B, Steele L, Heng M. High‐throughput process development for recombinant protein purification. Biotechnol Bioeng. 2006;93(4):618-30. [DOI:10.1002/bit.20702] [PMID]
3. Wood DW. New trends and affinity tag designs for recombinant protein purification. Curr Opin Struct Biol. 2014;26:54-61. [DOI:10.1016/j.sbi.2014.04.006] [PMID]
4. Urh M, Simpson D, Zhao K. Affinity chromatography: general methods. Methods Enzymol. 2009;463:417-38. [DOI:10.1016/S0076-6879(09)63026-3]
5. Hage DS, Anguizola JA, Bi C, Li R, Matsuda R, Papastavros E, et al. Pharmaceutical and biomedical applications of affinity chromatography: recent trends and developments. J Pharm Biomed Anal. 2012;69:93-105. [DOI:10.1016/j.jpba.2012.01.004] [PMID] [PMCID]
6. Behere K, Yoon S. Chromatography bioseparation technologies and in-silico modelings for continuous production of biotherapeutics. Journal of Chromatography. 2020;1627:461376. [DOI:10.1016/j.chroma.2020.461376] [PMID]
7. Lai W-J, Lin S-C. Hydroxyethyl cellulose-grafted loofa sponge-based metal affinity adsorbents for protein purification and enzyme immobilization. Process Biochemistry. 2018;74:141-147. [DOI:10.1016/j.procbio.2018.08.024]
8. Thakur VK, Thakur MK. Processing and characterization of natural cellulose fibers/thermoset polymer composites. Carbohydr polym. 2014;109:102-17. [DOI:10.1016/j.carbpol.2014.03.039] [PMID]
9. Ilyas R, Sapuan S, Sanyang ML, Ishak MR, Zainudin E. Nanocrystalline cellulose as reinforcement for polymeric matrix nanocomposites and its potential applications: a review. Current Analytical Chemistry. 2018;14(3):203-25. [DOI:10.2174/1573411013666171003155624]
10. Mishra S, Kharkar PS, Pethe AM. Biomass and waste materials as potential sources of nanocrystalline cellulose: Comparative review of preparation methods (2016-Till date). Carbohydr polym. 2019;207:418-27. [DOI:10.1016/j.carbpol.2018.12.004] [PMID]
11. Sidar A, Albuquerque ED, Voshol GP, Ram AF, Vijgenboom E, Punt PJ. Carbohydrate binding modules: diversity of domain architecture in amylases and cellulases from filamentous microorganisms. Front bioeng and biotechnol. 2020;8:871. [DOI:10.3389/fbioe.2020.00871] [PMID] [PMCID]
12. Armenta S, Moreno‐Mendieta S, Sánchez‐Cuapio Z, Sánchez S, Rodríguez‐Sanoja R. Advances in molecular engineering of carbohydrate‐binding modules. Proteins: Structure, Function, and Bioinformatics. 2017;85(9):1602-1617. [DOI:10.1002/prot.25327] [PMID]
13. Angelov A, Loderer C, Pompei S, Liebl W. Novel family of carbohydrate-binding modules revealed by the genome sequence of Spirochaeta thermophila DSM 6192. Appl Environ Microbiol. 2011;77(15):5483-9. [DOI:10.1128/AEM.00523-11] [PMID] [PMCID]
14. Campos BM, Liberato MV, Polikarpov I, Zeri ACdM, Squina FM. Cloning, purification, crystallization and preliminary X-ray studies of a carbohydrate-binding module from family 64 (StX). Acta Crystallogr F Struct Biol Commun. 2015;71(Pt 3):311-4. [DOI:10.1107/S2053230X15002198] [PMID] [PMCID]
15. Pires VM, Pereira PM, Brás JL, Correia M, Cardoso V, Bule P, et al. Stability and ligand promiscuity of type A carbohydrate-binding modules are illustrated by the structure of Spirochaeta thermophila StCBM64C. J Biol Chem. 2017;292(12):4847-4860. [DOI:10.1074/jbc.M116.767541] [PMID] [PMCID]
16. 16 Green MR, Hughes H, Sambrook J, MacCallum P. Molecular cloning: a laboratory manual. 2012. Cold Spring Harbor Laboratory Press, New York. 2012; 1890-92.
17. Chen Z, Zhao L, Ru J, Yu S, Yu H, Ren H, et al. A novel protein purification strategy mediated by the combination of CipA and Ssp DnaB intein. J Biotechnol. 2019;301:97-104. [DOI:10.1016/j.jbiotec.2019.06.002] [PMID]
18. Pfaunmiller EL, Paulemond ML, Dupper CM, Hage DS. Affinity monolith chromatography: a review of principles and recent analytical applications. Anal Bioanal Chem. 2013;405(7):2133-45. [DOI:10.1007/s00216-012-6568-4] [PMID] [PMCID]
19. Wan W, Wang D, Gao X, Hong J. Expression of family 3 cellulose-binding module (CBM3) as an affinity tag for recombinant proteins in yeast. Appl Microbiol Biotechnol. 2011;91(3):789-98. [DOI:10.1007/s00253-011-3373-5] [PMID]
20. Greenwood MJ, Ong E, Gilkes RN, Warren RAJ, Miller Jr CR, Kilburn GD. Cellulose-binding domains: potential for purification of complex proteins. Protein Eng. 1992;5(4):361-5. [DOI:10.1093/protein/5.4.361] [PMID]
21. Shpigel E, Goldlust A, Eshel A, Ber IK, Efroni G, Singer Y, et al. Expression, purification and applications of staphylococcal Protein A fused to cellulose‐binding domain. Biotechnol Appl Biochem. 2000;31(3):197-203. [DOI:10.1042/BA20000002] [PMID]
22. Liu Z, Pan Z, Koepsel R, Soong Y, Koepsel R, Ataai M, editors. A Novel Fusion Carbonic Anhydrase/cellulose Binding Protein For Capturing CO2. TheAIChE Annual Meeting; 2007.
23. Sugimoto N, Igarashi K, Samejima M. Cellulose affinity purification of fusion proteins tagged with fungal family 1 cellulose-binding domain. Protein Expr Purif. 2012;82(2):290-6. [DOI:10.1016/j.pep.2012.01.007] [PMID]
24. Ofir K, Berdichevsky Y, Benhar I, Azriel‐Rosenfeld R, Lamed R, Barak Y, et al. Versatile protein microarray based on carbohydrate‐binding modules. Proteomics. 2005;5(7):1806-14. [DOI:10.1002/pmic.200401078] [PMID]
25. Rodriguez B, Kavoosi M, Koska J, Creagh AL, Kilburn DG, Haynes CA. Inexpensive and generic affinity purification of recombinant proteins using a family 2a CBM fusion tag. Biotechnol Prog. 2004;20(5):1479-89. [DOI:10.1021/bp0341904] [PMID]
26. Xu Z, Bae W, Mulchandani A, Mehra RK, Chen W. Heavy metal removal by novel CBD-EC20 sorbents immobilized on cellulose. Biomacromolecules. 2002;3(3):462-5. [DOI:10.1021/bm015631f] [PMID]
27. Liao H, Myung S, Zhang Y-HP. One-step purification and immobilization of thermophilic polyphosphate glucokinase from Thermobifida fusca YX: glucose-6-phosphate generation without ATP. Appl Microbiol Biotechnol. 2012;93(3):1109-17. [DOI:10.1007/s00253-011-3458-1] [PMID]
28. Kavoosi M, Creagh AL, Turner RF, Kilburn DG, Haynes CA. Direct measurement of the kinetics of CBM9 fusion‐tag bioprocessing using luminescence resonance energy transfer. Biotechnol Prog. 2009;25(3):874-81. [DOI:10.1002/btpr.88] [PMID]

Add your comments about this article : Your username or Email:

Send email to the article author

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

© 2015 All Rights Reserved | Reports of Biochemistry and Molecular Biology

Designed & Developed by : Yektaweb