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The selective isolation of an active-site histidine peptide from chymotrypsin-alpha by diagonal peptide 'mapping'. An N-tau-carboxymethylhistidine diagonal peptide "mapping.".

ABSTRACT: 1. The selective isolation of an ;active' histidine peptide from reduced and cyanoethylated chymotrypsin-alpha inhibited with Tos-Phe-CH(2)Cl (l-1-tosylamido-2-phenylethyl chloromethyl ketone) was obtained with a His(tauCm) (N(tau)-carboxymethylhistidine) diagonal peptide-;mapping' technique. Performic acid vapours, used between the first and second dimensions of the diagonal peptide ;map', resulted in a peracid rearrangement of the alkylated (Tos-Phe-CH(2))-histidine-57 residue into an N(tau)-carboxymethylhistidine residue. The consequent change in electrophoretic mobility allowed isolation of peptides that contained the ;active' histidine. 2. Peptides containing methionine or S-cyanoethylcysteine were oxidized to their sulphones during the treatment. Peptides in which these residues were N-terminal were selectively isolated on the basis of the change in electrophoretic mobility at pH6.5 which was due to the depression of the pK of the terminal amino group by the inductive effect of the sulphonyl group. 3. An attempt to apply the method to subtilisin BPN' inhibited with l-1-benzyloxycarbonylamido-2-phenylethyl bromomethyl ketone failed to yield a peptide containing N(tau)-carboxymethylhistidine, although peptides containing N-terminal methionine were isolated by the procedure.

SUBMITTER: Stevenson KJ

PROVIDER: S-EPMC1166270 | biostudies-other | 1974 Apr

REPOSITORIES: biostudies-other

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Project description:Site-selective conjugation generally requires both (i) molecular engineering of the protein of interest to introduce a conjugation site at a defined location and (ii) a site-specific conjugation technology. Three N-terminal interferon α2-a (IFN) variants with truncated histidine tags were prepared and conjugation was examined using a bis-alkylation reagent, PEG(10kDa)-mono-sulfone 3. A histidine tag comprised of two histidines separated by a glycine (His2-tag) underwent PEGylation. Two more IFN variants were then prepared with the His2-tag engineered at different locations in IFN. Another IFN variant was prepared with the His-tag introduced in an α-helix, and required three contiguous histidines to ensure that two histidine residues in the correct conformation would be available for conjugation. Since histidine is a natural amino acid, routine methods of site-directed mutagenesis were used to generate the IFN variants from E. coli in soluble form at titres comparable to native IFN. PEGylation conversions ranged from 28-39%. A single step purification process gave essentially the pure PEG-IFN variant (>97% by RP-HPLC) in high recovery with isolated yields ranging from 21-33%. The level of retained bioactivity was strongly dependent on the site of PEG conjugation. The highest biological activity of 74% was retained for the PEG10-106(HGHG)-IFN variant which is unprecedented for a PEGylated IFN. The His2-tag at 106(HGHG)-IFN is engineered at the flexible loop most distant from IFN interaction with its dimeric receptor. The biological activity for the PEG10-5(HGH)-IFN variant was determined to be 17% which is comparable to other PEGylated IFN conjugates achieved at or near the N-terminus that have been previously described. The lowest retained activity (10%) was reported for PEG10-120(HHH)-IFN which was prepared as a negative control targeting a IFN site thought to be involved in receptor binding. The presence of two histidines as a His2-tag to generate a site-selective target for bis-alkylating PEGylation is a feasible approach for achieving site-selective PEGylation. The use of a His2-tag to strategically engineer a conjugation site in a protein location can result in maximising the retention of the biological activity following protein modification.

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The selective isolation of an active-site histidine peptide from chymotrypsin-alpha by diagonal peptide 'mapping'. An N-tau-carboxymethylhistidine diagonal peptide "mapping.".

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