Mycobacterium tuberculosis chaperonin 10 and N-truncated fragments: Their synthesis and purification by the isoelectric focusing technique carried out in solution

Pierlulgi Lucietto, Gianluca Fossati, Haydn L. Ball, Paola Giuliani, Paolo Mascagni

Research output: Contribution to journalArticle

6 Scopus citations

Abstract

The Mycobacterium tuberculosis chaperonin 10 protein and fragments corresponding to sequences 59-99, 51-99 and 26-99 were synthesised by the solid-phase methodology using a double coupling protocol and without the aid of capping agents. After the final acid cleavage using the low TFMSA-high HF protocol the polypeptides were purified by either the ion exchange chromatography/RP-HPLC combination or the isoelectric separation carried out in solution and followed by semi-preparative RP-HPLC. Comparison of the results obtained through the two approaches indicated that in general the isoeletricfocusing/HPLC combination was superior both in terms of recovery of final material and its purity. The advantages found were as follows: (i) Unlike ion exchange chromatography, no tailoring of the separation conditions is required. (ii) Several consecutive focusings can be carried out in progressively narrower pH gradients. This increases the separation resolution without the need of changing other separation parameters. (iii) Very little manipulation is needed, and each focusing requires 3-5 h. (iv) Full compatibility with non-ionic denaturants such as 8 M urea. This increases solubility so that using the ROTOFOR instrument described here 50-100 mg crude polypeptide can be processed daily. Thus the isoelectric focusing technique carried out in solution is a valid and inexpensive alternative to ion exchange chromatography.

Original languageEnglish (US)
Pages (from-to)308-323
Number of pages16
JournalJournal of Peptide Research
Volume49
Issue number4
StatePublished - Jul 15 1997

Keywords

  • Isoelectric focusing in solution
  • M. tuberculosis chaperonin 10
  • Peptide synthesis
  • Purification
  • ROTOFOR cell

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology

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