Abstract
An electrophoretic procedure based on isotachophoresis has been developed for protein purification on a preparative scale in the 10 to 500 mg range. The system is simple, uses well understood physical properties, does not need ampholyte spacers and is able to produce sterile products of clinical grade. We demonstrate the applicability of this apparatus for the purification of denatured recombinant proteins and complex mixtures of proteins. The system may also be used for both cationic and anionic purification of proteins in their native form. The system is scalable from analytical to preparative protein loads at consistently high protein yields and purity levels. Total protein loads may vary as much as 1000 fold with the use of interchangeable columns of varying diameter and constant length. At both preparative and analytical scales concentration of products at greater than 20 mg/ml are obtainable. Toxicological considerations are addressed with assays for endotoxin, acrylamide and SDS concentrations, as well as the prevention of covalent protein modification.
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Ridley, R.G., Takacs, B., Lahm, H., Delves, C.J., Goman, M., Certa, U., Matile, H., Woollett, G.R. and Scaife, J.G. 1990. Characterisation and sequence of a protective rhoptry antigen from Plasmodium falciparum. Mol. Biochem. Parasitol. 41: 125–134.
Saul, A., Cooper, J., Hauquitz, D., Irving, D., Cheng, Q., Stowers, A. and Limpaiboon, T. 1992. The 42-kilodalton rhoptry-associated protein of Plasmodium falciparum. Mol. Biochem. Parasitol. 50: 139–150.
Ridley, R.G., Takacs, B., Etlinger, H. and Scaife, J.G. 1990. A rhoptry antigen of Plasmodium falciparum is protective in Saimiri monkeys. Parasitology 101: 187–192.
Baumann, G. and Chrambach, A. 1976. Gram-preparative protein fractionation by isotachophoresis: Isolation of human growth hormone isohormones. Proc. Nat. Acad. Sci. USA 73: 732–736.
Holloway, C.J. and Battersby, R.V. 1984. Preparative Isotachophoresis. Methods Enzymol. 104: 281–301.
Stowers, A., Prescott, N., Cooper, J., Takacs, B., Stueber, D., Kennedy, P. and Saul, A. 1995. Immunogenicity of recombinant Plasmodium falciparum rhoptry associated proteins 1 and 2. Parasit. Immunol. In press.
Righetti, P.G., Faupel, M. and Wenisch, E. 1992. Preparative electrophoresis with and without immobilized pH gradients, p. 159–200. In: Advances in Electrophoresis. Chrambach, A., Dunn, M. J. and Radola, B. J. (Eds.). VCH Publishers, New York, NY.
Moos, M., Nguyen, N.Y. and Liu, T.-Y. 1988. Reproducable high yield sequencing of proteins electrophoretically separated and transferred to an inert support. J. Biol. Chem. 263: 6005–6008.
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of the bacteriophage T4. Nature 277: 680–682.
Waite, J.H. and Wang, C.-Y. 1975. Spectrophotometric measurement of dodecyl sulfate with basic fuchsin. Anal. Biochem. 70: 279–280.
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Stowers, A., Spring, K. & Saul, A. Preparative Scale Purification of Recombinant Proteins to Clinical Grade by Isotachophoresis. Nat Biotechnol 13, 1498–1503 (1995). https://doi.org/10.1038/nbt1295-1498
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DOI: https://doi.org/10.1038/nbt1295-1498