Affinity purification of 101 residue rat cpn10 using a reversible biotinylated probe.
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.
Ball
H L
HL
Italfarmaco Research Centre, Milan, Italy.
Bertolini
G
G
Mascagni
P
P
D000818:Animals; D001360:Avidin; D001710:Biotin; D018835:Chaperonin 10 / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D007202:Indicators and Reagents; D015335:Molecular Probes; D008970:Molecular Weight; D051381:Rats
D018835:Chaperonin 10; D007202:Indicators and Reagents; D015335:Molecular Probes; D001360:Avidin; D001710:Biotin