Enzymes, purification by affinity chromatography

Cuatrecasas, P., Wilchek, M., Anfinsen, C. B. (1968). Selective enzyme purification by affinity chromatography. Proc. Natl. Acad. Sci. USA, 61, 636-643. 

Cuatrecasas P, Wilchek M, and Anfinsen CB (1968) Selective enzyme purification by affinity chromatography. Proceedings of the National Academy of Sciences of the USA 61 636-643. 

A procedure for enzyme purification by affinity chromatography using a non-covalently bound absorbent has been described with particular reference to lipophilic interactions. ... 

Purification of peptides by immobilized enzymes is a relatively recent approach that may have a potential use in the purification of genetically engineered proteins. Purification of recombinant proteins is often difficult and involves many purification steps for a very low yield. Smith et al. found that a specific metal-chelating peptide on the NH2 terminus of a protein can be used to purify that protein with immobilized ion affinity chromatography. The nucleotide sequence that codes for the expressed protein is extended to include codons for the chelating peptide. This concept of amino acid addition to recombinant proteins can also be beneficial in protein purification by affinity chromatography with immobilized enzymes. 

Wikstrom, P. and Larsson, P.-O., Affinity fibre—A new support for rapid enzyme purification by high-performance liquid affinity chromatography, J. Chromatogr., 388, 123-134, 1987. 

With the advent of affinity chromatography to isolate enzymes [7] it was possible to adapt the method for the isolation of antibodies [8], The purification of many types of anti-carbohydrate antibodies with specificity for carbohydrate residues of macromolecules has been achieved. The antibodies were isolated by affinity chromatography on adsorbents with proper ligands. The immunodeterminants of the antigens have been found to be mono- or disaccharide residues of the antigen. The anti-carbohydrate antibodies with unique properties are useful in a variety of medicinal and technological applications. In this article the emphasis is on polyclonal antibodies specifically the preparation, properties and uses of anti-carbohydrate antibodies. 

The purification was by chromatography on DEAE cellulose. Electrophoresis of the purified enzyme and the original filtrate are shown in Fig. (27) [71]. Rabbits were immunized with the pure glucoamylase [72]. Antibodies in the serum were purified by affinity chromatography. The affinity pattern for glucoamylase purification is shown in Fig. (28). Electrofocusing and assay by the coupled method for antibodies is shown in Fig. (29). The glucoamylase antibodies consist of 8 isomers [73],... 

Ogunyemi, E.O., Pittner, F., and Hoffmannostenhof, O., 1978, Studies on biosynthesis of cyclitols 36. Purification of myo-inositol-1-phosphate synthase of duckweed, Lemna-Gibba, to homogeneity by affinity chromatography on nad-sepharose molecular and catalytic properties of enzyme. Hoppe-Seylers Zeitschrift Fur Physiologische Chemie 359 613-616. 

Lee CY, Johansson CJ. Purification of cofactor-dependent enzymes by affinity chromatography. Analyt. Biochem. 1977 77 90-102. 

Trypsin and chymotrypsin. Purification of both trypsin and chymo-trypsin by affinity chromatography is now a routine tool in many laboratories. Most commercial trypsin preparations are contaminated with trace amounts of chymotrypsin which restricts the use of the enzyme in protein structural studies. However, the soya trypsin inhibitor-agarose media provides a convenient column for separation of the enzymes. 

Fig. 1 Schematic illustration of purification of a peptide by affinity chromatography on an immobilized enzyme.

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