Journal
CHEMISTRY & BIOLOGY
Volume 8, Issue 7, Pages 673-679Publisher
CURRENT BIOLOGY LTD
DOI: 10.1016/S1074-5521(01)00044-8
Keywords
covalent capture; polypeptide purification; recombinant protein purification
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Background: Purification of polypeptides and proteins derived from recombinant DNA techniques and of long synthetic polypeptides often represents a challenge, Affinity methods exist, but generally require addition of a large recognition unit to the target protein and use of expensive purification media. Use of large units is dictated by the characteristics of non-covalent complexes, where the energy necessary to form the complex derives from the sum of multiple weak energy interactions. Covalent interactions in contrast are of high energy, even when only a few bonds are formed. We decided to explore the use of the reversible covalent bond formed between N-terminal cysteine and threonine residues with an aldehyde as a method of protein purification. Results: A series of test peptides with N-terminal cysteine and threonine were captured by a polyethyleneglycol-polyacrylamide resin to which an aldehyde function had been grafted. Peptides with other amino acids at the N-terminus did not interact with the resin. A recombinant polypeptide with N-terminal cysteine, was purified to 90% purity in one step. Polypeptides were eluted from the resin simply by adding a hydroxylamine derivative, which reacts with aldehyde functions to form an oxime. Conclusions: Polypeptides possessing N-terminal cysteine or threonine can be easily purified using this 'covalent capture' approach. (C) 2001 Elsevier Science Ltd. All rights reserved.
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