Covalent capture: a new tool for the purification of synthetic and recombinant polypeptides.
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.
Villain
M
M
Department of Medical Biochemistry, University Medical Center, Rue Michel-Servet 1, CH-1211 4, Geneva, Switzerland.
Vizzavona
J
J
Rose
K
K
D000447:Aldehydes / Q000737:chemistry; D018946:Chemokine CCL5 / Q000138:chemical synthesis / Q000302:isolation & purification; D002845:Chromatography / Q000379:methods / Q000592:standards; D002851:Chromatography, High Pressure Liquid; D008722:Methods; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000302:isolation & purification; D010455:Peptides / Q000138:chemical synthesis / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification; D012116:Resins, Plant / Q000737:chemistry*
D000447:Aldehydes; D018946:Chemokine CCL5; D010446:Peptide Fragments; D010455:Peptides; D011994:Recombinant Proteins; D012116:Resins, Plant