On-resin biotinylation of chemically synthesized proteins for one-step purification.
A general, convenient, one-step purification procedure for chemically synthesized proteins present in low yields using on-resin biotinylation is reported. The protein, terminally deprotected and neutralized on-resin, is stirred in dimethylformamide and then biotinylated with N-hydroxysuccinimidobiotin (2 mg/mg protein on-resin) for 24 h at 45 degrees C. Following low/high hydrogen fluoride cleavage (J. P. Tam, W. F. Heath, and R. B. Merrifield (1983) J. Amer. Chem. Soc. 105, 6442-6455) the crude cleavage product was applied to an avidin agarose column. The column was washed with phosphate-buffered saline until all unbound materials had been eluted off. Then the biotinylated protein was eluted with 0.1 M glycine HCl, pH 2.0. A pilot experiment with two unrelated peptides on-resin established the experimental conditions for biotinylation. We then demonstrated that the chemically synthesized 153 residue [Asp205]-interleukin-1 beta (117-269), present in less than 1% yield in the crude HF cleavage mixture, could be purified to homogeneity in one step. In addition 70 and 114 residue synthetic fragments, (200-269) and (156-269), were also purified in this manner. Biotinylation on-resin appears to be an attractive method of purifying low yield chemically synthesized proteins and for preparing proteins with biotinyl moieties at specific locations such as the amino terminus.
Lobl
T J
TJ
Biopolymer Chemistry, Upjohn Company, Kalamazoo, Michigan 49001.
Deibel
M R
MR
Yem
A W
AW
D001360:Avidin; D001710:Biotin; D002847:Chromatography, Agarose; D003101:Collodion; D004591:Electrophoresis, Polyacrylamide Gel; D004798:Enzymes / Q000302:isolation & purification; D007375:Interleukin-1 / Q000302:isolation & purification; D010455:Peptides / Q000302:isolation & purification; D011506:Proteins / Q000138:chemical synthesis / Q000302:isolation & purification*; D012116:Resins, Plant
D004798:Enzymes; D007375:Interleukin-1; D010455:Peptides; D011506:Proteins; D012116:Resins, Plant; D001360:Avidin; D001710:Biotin; D003101:Collodion