Enzymes: Purification


Purification is the first and most critical step in the study of a protein or enzyme: most modern analytical techniques do not tolerate impurities and some also demand large amounts of material. Each protein is different, even homologues from different sources, and is likely to require a unique purification scheme.

Keywords: protein; chromatography; extract; fusion protein

Figure 1.

Elution of protein from an anion exchange column. Beads are shown in blue, protein in red; buffer ions are white circles. The protein is passed over the column and binds. Then an increasing salt gradient is passed over the column, displacing the protein.

Figure 2.

The charge of a protein determines the type of ion exchange resin to be used.

Figure 3.

Proteins eluting later from a gel filtration column become diluted. The figure shows an idealized chromatogram illustrating equimolar amounts of three proteins eluting at different times from a gel exclusion matrix. Note that the later peaks are broader and not as intense. Proteins eluting earlier show sharper peaks.


Further Reading

Ausubel FM, Brent R, Kingston RE et al. (eds) (1988) Current Protocols in Molecular Biology. New York: Wiley.

Avarbock D, Salem J, Li LS, Wang ZM and Rubin H (1999) Cloning and characterization of a bifunctional RelA/SpoT homologue from Mycobacterium tuberculosis. Gene 233(1–2): 261–269.

Scopes RK (1993) Protein Purification: Principles and Practice, 3rd edn. New York: Springer‐Verlag.

Wang Z, Walter M, Selwood T, Rubin H and Schechter NM (1998) Recombinant expression of human mast cell proteases chymase and tryptase. Biological Chemistry 379(2): 167–174.

Wilchek M and Chaiken I (2000) An overview of affinity chromatography. Methods in Molecular Biology 147: 1–6.

Winzor DJ (2000) From gel filtration to biosensor technology: the development of chromatography for the characterization of protein interactions. Journal of Molecular Recognition 13(5): 279–298.

Yang F, Lu G and Rubin H (1994) Isolation of ribonucleotide reductase from Mycobacterium tuberculosis and cloning, expression, and purification of the large subunit. Journal of Bacteriology 176(21): 6738–6743.

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Salem, Jerome(Apr 2001) Enzymes: Purification. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0000863]