TY - JOUR
T1 - Mycobacterium tuberculosis chaperonin 10 and N-truncated fragments
T2 - Their synthesis and purification by the isoelectric focusing technique carried out in solution
AU - Lucietto, Pierlulgi
AU - Fossati, Gianluca
AU - Ball, Haydn L.
AU - Giuliani, Paola
AU - Mascagni, Paolo
PY - 1997
Y1 - 1997
N2 - 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.
AB - 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.
KW - Isoelectric focusing in solution
KW - M. tuberculosis chaperonin 10
KW - Peptide synthesis
KW - Purification
KW - ROTOFOR cell
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U2 - 10.1111/j.1399-3011.1997.tb01131.x
DO - 10.1111/j.1399-3011.1997.tb01131.x
M3 - Article
C2 - 9176814
AN - SCOPUS:0031001088
SN - 1397-002X
VL - 49
SP - 308
EP - 323
JO - Journal of Peptide Research
JF - Journal of Peptide Research
IS - 4
ER -