Affinity purification of synthetic peptides.

Abstract

A general strategy and a specific tactic for affinity purification of polypeptides synthesized on solid supports are desbribed and demonstrated. The desired peptide chains were distinguished from terminated peptide chains before removal from the support by attachment of an affinity reagnet (cysteinyl-methionine) bearing an affinity group (thiol) and a binding group (carboxylic acid). After cleavage from the synthetic support, the affinity-labeled peptides (Cys-Met-peptides) were bound to an affinity receptor (organomercurial-agarose) and thus separated from terminated peptides and all other peptides lacking the affinity group. The desired synthetic peptide was obtained by separation of the affinity reagent (loss of Cys-Met by cyanogen bromide cleavage). This general affinity purification strategy is independent of the length or amino acid sequence of the desired peptide. After assembly of ribonuclease-(111-124)-tetradecapeptide, using radiolabeled acetic anhudride for termination of uncoupled in termediates, essentially all (greater than 98.5%) of the acetylated delection peptides were removed by employing the organomercurial Cys-Met tactic. Similarly, the purity of crude synthetic histone H4-(1-37)-heptatriacontapeptide was increased six-fold by using this tactic to remove terminated peptides. A related dimeric Cys-Met tactic is outlined for affinity purification of peptides containing internal cysteine and methionine residues.

Use of synthetic peptides to identify an N-terminal epitope on mouse gamma interferon that may be involved in function.

“…IFN-'y-(1-10) showed a single peak on HPLC after the desalting procedure and, thus, did not require thiol-affinity purification. IFN-y-(10-30) and IFN-y-(21-44) required partial purification by thiol-affinity chromatography (8) characteristic peaks upon reinjection on HPLC, whereas IFN-y-(21-44) showed two peaks. These two peaks demonstrated identical amino acid composition and probably differed only in the oxidation state of their a-cysteines, since only the peak eluted earlier gave a positive reaction for sulfhydryl groups with Ellman's reagent (10).…”

“…Its physicochemical and immunochemical properties have been described (3). A cysteine residue was added at the N-terminal position of IFN-y-(1-10), (IFN-y- , and IFN--y-(21-44) to facilitate purification by thiolaffinity chromatography (8). Peptides were synthesized on an Applied Biosystems (model 430A) automated peptide synthesizer with standard software programs.…”

Formation of a covalent Hg‐Cys‐bond during mercurial activation of PMNL procollagenase gives evidence of a cysteine‐switch mechanism

“…300 pl Af'n-gel-50l [14] were washed several time~ with buffer [21) mM Tris-HCl pH 7.5; 10 mM CaCI_,; 0.0005% Triton X-100). 500/~1 procollagena~ (150 ~g) in tn~' gtme buffer was sulajeered to tile gei material and continuously shaken nl 37°C.…”

Purification of synthetic peptides Immobilized metal ion affinity chromatography (IMAC)*

“…The mercapto function has already been exploited for chromatographic purification of cysteine-containing peptides by reaction with immobilized mercury derivatives (2) or activated thiols (3). The mercapto function has already been exploited for chromatographic purification of cysteine-containing peptides by reaction with immobilized mercury derivatives (2) or activated thiols (3).…”