Matteo Villain

Covalent capture: a new tool for the purification of synthetic and recombinant polypeptides

BACKGROUNDnPurification 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.nnnRESULTSnA 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.nnnCONCLUSIONSnPolypeptides possessing N-terminal cysteine or threonine can be easily purified using this covalent capture approach.

Covalent capture purification of polypeptides after SPPS via a linker removable under very mild conditions

The covalent purification of polypeptides possessing an N-terminal cysteine or threonine residue via formation of a thiazolidine or oxazolidine with an aldehyde-functionalized-resin has been successfully demonstrated. To extend the applicability of this approach to any possible N-terminal residue, a special linker derived from (S)-4-amino-2-hydroxy-butyric acid was incorporated into peptidyl-resin. This linker represents the connecting point between the capture unit (cysteine) useful for the isolation of the desired polypeptide and the desired N-terminus. The target polypeptide was recovered by periodate oxidation, which cleaved the covalent bond between the linker and the last residue of polypeptide under very mild conditions.

Chemical Ligation of Multiple Peptide Fragments Using a New Protection Strategy

Synthesis of large polypeptides requires the assembly by chemical ligation [1] of three or more unprotected peptide segments of thirty or more amino acids. In the synthesis in the C-to-N direction, a key issue is the potential reactivity of the middle segments, which would bear both an N-terminal Cys and C-terminal thioester. Up until now, an Acm [2] or Msc [3] group has usually been employed to temporarily protect either the Ss or Nα of the N-terminal Cys residue, to prevent self condensation and other side reactions. In order to overcome the limitations of these two approaches, we investigated the use of a thiazolidine derivative, thioproline, as a precursor of the N-terminal Cys in the synthesis of the middle peptide segments.