Abstract
Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90 %. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Ervin MA, Luss D (1970) Effect of fouling on stability of adiabatic packed bed reactors. AlChE J 16:979–984
Siu S, Baldascini H, Hearle D, Hoare M, Tichener-Hooker NJ (2006) Effect of fouling on the capacity and breakthrough characteristics of a packed bed ion exchange chromatography column. Bioprocess Biosyst Eng 28:405–414
Bibi NS, Gavara PR, Espinosa SLS, Grasselli M, Fernández-Lahore M (2011) Synthesis and performance of 3D-megaporous structures for enzyme immobilization and protein capture. Biotechnol Prog 27:1329–1338
Hedström M, Plieva F, Galaev IY, Mattiasson B (2008) Monolithic macroporous albumin/chitosan cryogel structure: a new matrix for enzyme immobilization. Anal Bioanal Chem 390:907–912
Lozinsky VI (2008) Polymeric cryogels as a new family of macroporous and supermacroporous materials for biotechnological purposes. Russ Chem Bull 57:1015–1032
Plieva FM, Galaev IY, Mattiasson B (2007) Macroporous gels prepared at subzero temperatures as novel materials for chromatography of particulate-containing fluids and cell culture applications. J Sep Sci 30:1657–1671
Plieva FM, Galaev IY, Noppe W, Mattiasson B (2008) Cryogel applications in microbiology. Trends Microbiol 16:543–551
Plieva FM, Kirsebom H, Mattiasson B (2011) Preparation of macroporous cryostructurated gel monoliths, their characterization and main applications. J Sep Sci 34:2164–2172
Arvidsson P, Plieva FM, Savina IN, Lozinsky VI, Fexby S, Bulow L, Galaev IY, Mattiasson B (2002) Chromatography of microbial cells using continuous supermacroporous affinity and ion-exchange columns. J Chromatogr A 977:27–38
Du K-F, Yang D, Sun Y (2007) Fabrication of high-permeability and high-capacity monolith for protein chromatography. J Chromatogr A 1163:212–218
Yao K, Shen S, Yun J, Wang LH, He XJ, Yu XM (2006) Preparation of polyacrylamide-based supermacroporous monolithic cryogel beds under freezing-temperature variation conditions. Chem Eng Sci 61:6701–6708
Yao K, Yun J, Shen S, Chen F (2007) In-situ graft-polymerization preparation of cation-exchange supermacroporous cryogel with sulfo groups in glass columns. J Chromatogr A 1157:246–251
Yao K, Yun J, Shen S, Wang LH, He XJ, Yu XM (2006) Characterization of a novel continuous supermacroporous monolithic cryogel embedded with nanoparticles for protein chromatography. J Chromatogr A 1109:103–110
Arvidsson P, Plieva FM, Lozinsky VI, Galaev IV, Mattiasson B (2003) Direct chromatographic capture of enzyme from crude homogenate using immobilized metal affinity chromatography on a continuous supermacroporous adsorbent. J Chromatogr A 986:275–290
Chaga GS (2001) Twenty-five years of immobilized metal ion affinity chromatography: past, present and future. J Biochem Biophys Methods 49:313–334
Gutiérrez R, Martín del Valle EM, Galán MA (2007) Immobilized metal‐ion affinity chromatography: status and trends. Sep Purif Rev 36:71–111
Porath J (1992) Immobilized metal ion affinity chromatography. Protein Express Purif 3:263–281
Arnold FH (1991) Metal-affinity separations: a new dimension in protein processing. Nat Biotechnol 9:151–156
Pearson RG (1990) Hard and soft acids and bases – the evolution of a chemical concept. Coord Chem Rev 100:403–425
Bibi NS, Singh NK, Dsouza RN, Aasim M, Fernández-Lahore M (2013) Synthesis and performance of megaporous immobilized metal-ion affinity cryogels for recombinant protein capture and purification. J Chromatogr A 1272:145–149
Rohde BH, Schmid R, Ullrich MS (1999) Thermoregulated expression and characterization of an NAD(P)H-dependent 2-cyclohexen-1-one reductase in the plant pathogenic bacterium Pseudomonas syringae pv. glycinea. J Bacteriol 181:814–822
Levitzki A, Pecht I, Berger A (1972) Copper-poly-L-histidine complexes. II. Physicochemical properties. J Am Chem Soc 94:6844–6849
Gallagher SR (2008) SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Current protocols essential laboratory techniques. Wiley, Hoboken, NJ, pp 7.3.1–7.3.25
Kruger N (1994) The Bradford Method for Protein Quantitation. In: Walker J (ed) Basic protein and peptide protocols. Methods in molecular biology™, vol 32. Humana, Totowa, NJ, pp 9–15
Gibbins J (2004) Techniques for Analysis of Proteins by SDS-Polyacrylamide Gel Electrophoresis and Western Blotting. In: Gibbins J, Mahaut-Smith M (eds) Platelets and megakaryocytes. Methods in molecular biology™, vol 273. Humana, Totowa, NJ, pp 139–151
D'Souza F, Lali A (1999) Purification of papain by immobilized metal affinity chromatography (IMAC) on chelating carboxymethyl cellulose. Biotechnol Tech 13:59–63
Singh NK, Dsouza RN, Grasselli M, FernÃndez-Lahore M (2013) High capacity cryogel-type adsorbents for protein purification. J Chromatogr A 1355:143–148
Acknowledgements
The authors thank Prof. Matthias S. Ullrich for providing cells carrying the Ncr gene for the production of the His6-tagged protein. M.F.L. is a member of the National Council for Science and Technology, Buenos Aires, Argentina. This work was funded by the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 312004.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Singh, N.K., DSouza, R.N., Bibi, N.S., Fernández-Lahore, M. (2015). Direct Capture of His6-Tagged Proteins Using Megaporous Cryogels Developed for Metal-Ion Affinity Chromatography. In: Reichelt, S. (eds) Affinity Chromatography. Methods in Molecular Biology, vol 1286. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2447-9_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2447-9_16
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2446-2
Online ISBN: 978-1-4939-2447-9
eBook Packages: Springer Protocols