Affinity purification of 101 residue rat cpn10 using a reversible biotinylated probe.

H. L. Ball, G. Bertolini, P. Mascagni

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

The purification of large synthetic peptides using conventional separation techniques often results in poor yields and homogeneity due to the accumulation of chromatographically similar deletion and truncated impurities. We have developed a highly effective synthetic strategy and one-step purification procedure that is based on (i) the application of single coupling using HBTU/HOBt activation to reduce incomplete couplings, (ii) the use of N-(2-chlorobenzyloxycarbonyloxy)succinimide as a capping agent to terminate deletion sequences and (iii) the N-terminal derivatization of the complete peptidyl-resin with a reversible Fmoc-based chromatographic probe possessing enhanced physico-chemical properties (i.e. hydrophobicity, charge or affinity label). We report the application of a biotinylated probe, activated as the succinimidyl carbonate, for the purification of a 101 residue chaperonin protein from Rattus norvegicus (rat cpn10), previously synthesized using an optimized synthetic protocol. Biotinylated rat cpn10 was separated from underivatized impurities on an immobilized monomeric avidin column. Free rat cpn10 was released from avidin-agarose column with 5% aqueous triethylamine and after desalting by RP-HPLC gave 9.9% recovery. Characterization and assessment of homogeneity was achieved using ESI-MS, CZE and RP-HPLC.

Original languageEnglish (US)
Pages (from-to)288-294
Number of pages7
JournalJournal of peptide science : an official publication of the European Peptide Society
Volume1
Issue number5
StatePublished - Sep 1995

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Purification
Rats
Affinity Labels
Impurities
Chaperonins
Salt removal
Avidin
High Pressure Liquid Chromatography
Hydrophobicity
Chemical properties
Sequence Deletion
Resins
Chemical activation
Hydrophobic and Hydrophilic Interactions
Recovery
Peptides
Proteins
succinimidyl carbonate
succinimide
1-hydroxybenzotriazole

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry

Cite this

Affinity purification of 101 residue rat cpn10 using a reversible biotinylated probe. / Ball, H. L.; Bertolini, G.; Mascagni, P.

In: Journal of peptide science : an official publication of the European Peptide Society, Vol. 1, No. 5, 09.1995, p. 288-294.

Research output: Contribution to journalArticle

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