Abstract
A positively charged protein domain, denoted Zbasic, can be used as a general purification tag for purification of recombinantly produced target proteins by cation-exchange chromatography. The Zbasic domain is constructed from the Protein A-derived Z-domain, and engineered to be highly charged, which allows selective capture on a cation exchanger at physiological pH values. Moreover, Zbasic is selective also under denaturing conditions and can be used for purification of proteins solubilized from inclusion bodies. Zbasic can then be used as a flexible linker to the cation-exchanger resin, and thereby allows solid-phase refolding of the target protein.
Herein, protocols for purification of soluble Zbasic-tagged fusion proteins , as well as for integrated purification and solid-phase refolding of insoluble fusion proteins , are described. In addition, a procedure for enzymatic tag removal and recovery of native target protein is outlined.
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References
Asplund M, Ramberg M, Johansson B-L (2000) Development of a cleaning in place protocol and repetitive application of Escherichia coli homogenate on STREAMLINE™ Q XL. Process Biochem 35:1111–1118
Hale G, Drumm A, Harrison P et al (1994) Repeated cleaning of protein A affinity column with sodium hydroxide. J Immunol Methods 171:15–21
Anspach FB, Curbelo D, Hartmann R et al (1999) Expanded-bed chromatography in primary protein purification. J Chromatogr A 865:129–144
Feuser J, Walter J, Kula MR et al (1999) Cell/adsorbent interactions in expanded bed adsorption of proteins. Bioseparation 8:99–109
Hedhammar M, Gräslund T, Hober S (2005) Protein engineering strategies for selective protein purification. Chem Eng Technol 28:1315–1325
Nilsson B, Moks T, Jansson B et al (1987) A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng 1:107–113
Graslund T, Lundin G, Uhlen M et al (2000) Charge engineering of a protein domain to allow efficient ion-exchange recovery. Protein Eng 13:703–709
Link AJ, Robison K, Church GM (1997) Comparing the predicted and observed properties of proteins encoded in the genome of Escherichia coli K-12. Electrophoresis 18:1259–1313
Graslund T, Ehn M, Lundin G et al (2002) Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner. J Chromatogr A 942:157–166
Hedhammar M, Alm T, Graslund T et al (2006) Single-step recovery and solid-phase refolding of inclusion body proteins using a polycationic purification tag. Biotechnol J 1:187–196
Wiesbauer J, Bolivar JM, Mueller M et al (2011) Oriented immobilization of enzymes made fit for applied biocatalysis: non-covalent attachment to anionic supports using Zbasic2 module. ChemCatChem 3:1299–1303
Bolivar JM, Nidetzky B (2012) Oriented and selective enzyme immobilization on functionalized silica carrier using the cationic binding module Z basic2: design of a heterogeneous D-amino acid oxidase catalyst on porous glass. Biotechnol Bioeng 109:1490–1498
Bolivar JM, Nidetzky B (2012) Positively charged mini-protein Z basic2 as a highly efficient silica binding module: opportunities for enzyme immobilization on unmodified silica supports. Langmuir 28:10040–10049
Bolivar JM, Luley-Goedl C, Leitner E et al (2017) Production of glucosyl glycerol by immobilized sucrose phosphorylase: options for enzyme fixation on a solid support and application in microscale flow format. J Biotechnol 257:131–138
Hedhammar M, Jung HR, Hober S (2006) Enzymatic cleavage of fusion proteins using immobilised protease 3C. Protein Expr Purif 47:422–426
Shi S, Chen H, Jiang H et al (2017) A novel self-cleavable tag Zbasic–ΔI-CM and its application in the soluble expression of recombinant human interleukin-15 in Escherichia coli. Appl Microbiol Biotechnol 101:1133–1142
Yip TT, Nakagawa Y, Porath J (1989) Evaluation of the interaction of peptides with Cu(II), Ni(II), and Zn(II) by high-performance immobilized metal ion affinity chromatography. Anal Biochem 183:159–171
Hedhammar M, Stenvall M, Lonneborg R et al (2005) A novel flow cytometry-based method for analysis of expression levels in Escherichia coli, giving information about precipitated and soluble protein. J Biotechnol 119:133–146
Alm T, Steen J, Ottosson J et al (2007) High-throughput protein purification under denaturating conditions by the use of cation exchange chromatography. Biotechnol J 2:709–716
Graslund T, Hedhammar M, Uhlen M et al (2002) Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology. J Biotechnol 96:93–102
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Hedhammar, M., Nilvebrant, J., Hober, S. (2021). Zbasic: A Purification Tag for Selective Ion-Exchange Recovery. In: Labrou, N.E. (eds) Protein Downstream Processing. Methods in Molecular Biology, vol 2178. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0775-6_12
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DOI: https://doi.org/10.1007/978-1-0716-0775-6_12
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