TY - JOUR
T1 - Chemical synthesis and purification of proteins
T2 - A methodology
AU - Ball, Haydn L.
AU - Mascagni, Paolo
PY - 1996
Y1 - 1996
N2 - 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.
AB - 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.
KW - Capping
KW - Chaperonin 10 from Rattus norvegicus
KW - Fluorenylmethoxycarbonyl
KW - Peptide synthesis
KW - Purification
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U2 - 10.1111/j.1399-3011.1996.tb01104.x
DO - 10.1111/j.1399-3011.1996.tb01104.x
M3 - Article
C2 - 8844261
AN - SCOPUS:0029773396
SN - 0367-8377
VL - 48
SP - 31
EP - 47
JO - International Journal of Peptide and Protein Research
JF - International Journal of Peptide and Protein Research
IS - 1
ER -