AFFINITY PURIFICATION OF PROTEINS USING LIGANDS DERIVED FROM PEPTIDE LIBRARIES

Peptide libraries can be used to identify ligands that bind specifical ly to a desired protein. These peptides may have significant advantage s as specific ligands for affinity chromatography separations. This ar ticle describes the use of one of such peptide, Try-Asn-Phe-Glu-Val-Le u, as a ligand for the purification of S-protein using affinity chroma tography, General strategies for peptide immobilization are discussed and the conditions for peptide immobilization to Emphaze(TM) gel are o ptimized. The effects of peptide orientation and peptide densities on protein binding are studied. Results indicate that the peptide affinit y is not affected by the orientation of the peptide during immobilizat ion, but association constants can be reduced by one order of magnitud e when compared with the values in solution, With increased peptide de nsity, the protein binding capacity of the gel increases, but both the percentage of peptide utilization and apparent binding constant betwe en immobilized peptide and S-protein decrease. S-protein is separated from a mixture with BSA via affinity chromatography using specific elu tion with the peptide in solution. Finally, direct purification of S-p rotein from an enzymatic digestion mixture of ribonuclease A is demons trated. (C) 1995 John Wiley and Sons, Inc.