Chemical synthesis and purification of proteins: A methodology

Haydn L. Ball, Paolo Mascagni

Research output: Contribution to journalArticle

32 Scopus citations

Abstract

Classical stepwise solid-phase peptide synthesis (SPPS) has been used successfully for the synthesis of proteins up to 150 residues in length, although usually with poor yields and homogeneity. The major limitation has been the inability to separate chromatographically similar deletion and truncated impurities from the target sequence. We have developed a highly effective protocol for stepwise SPPS and 'one-step' purification of small proteins. To demonstrate the effectiveness of the methodology we synthesised the 101 residue chaperonin 10 protein from Rattus norvegicus (Rat Cpn10) using three different chemical protocols. Highly homogeneous Rat Cpn10 was obtained using an optimised synthetic strategy and one-step purification procedure (method C), involving (i) HBTU/HOBt activation, (ii) N-(2-chlorobenzyloxycarbonyloxy)succinimide as capping agent and (iii) the incorporation of a reversible Fmoc-based chromatographic probe, derivatised with a lipophilic group for fast one-step RP purification, to give an overall yield of 9.6%. Analysis by ESI-MS indicated that the product was virtually free of deletion impurities, while RP-HPLC under four different conditions and CZE indicated that the protein was 100 and 84% pure, respectively. The spontaneous folding of Rat Cpn10 into its biologically active form was found to correlate well with the degree of purity as assessed by chromatography, ESI-MS and sequencing, since 29 (A), 55 (B) and 81% (C) of correctly folded heptameric structure was obtained. The degree of homogeneity was also reflected in the ability of purified Rat Cpn10 to facilitate the refolding of yeast enolase.

Original languageEnglish (US)
Pages (from-to)31-47
Number of pages17
JournalInternational Journal of Peptide and Protein Research
Volume48
Issue number1
StatePublished - Sep 5 1996

    Fingerprint

Keywords

  • Capping
  • Chaperonin 10 from Rattus norvegicus
  • Fluorenylmethoxycarbonyl
  • Peptide synthesis
  • Purification

ASJC Scopus subject areas

  • Biochemistry

Cite this