Affinity purification of proteins using ligands derived from peptide libraries

Abstract

Peptide libraries can be used to identify ligands that bind specifically to a desired protein. These peptides may have significant advantages as specific ligands for affinity chromatography separations. This article describes the use of one of such peptide, Try-Asn-Phe-Glu-Val-Leu, as a ligand for the purification of S-protein using affinity chromatography. General strategies for peptide immobilization are discussed and the conditions for peptide immobilization to Emphazetrade mark gel are optimized. The effects of peptide orientation and peptide densities on protein binding are studied. Results indicate that the peptide affinity is not affected by the orientation of the peptide during immobilization, but association constants can be reduced by one order of magnitude when compared with the values in solution.With increased peptide density, the protein binding capacity of the gel increases, but both the percentage of peptide utilization and apparent binding constant between immobilized peptide and S-protein decrease. S-protein is separated from a mixture with BSA via affinity chromatography using specific elution with the peptide in solution.Finally, direct purification of S-protein from an enzymatic digestion mixture of ribonuclease A is demonstrated.(c) 1995 John Wiley & Sons, Inc.

A simple protocol for combinatorial cyclic depsipeptide libraries sequencing by matrix-assisted laser desorption/ionisation mass spectrometry

“…Released peptides were eluted with 10 μl AcOH/CH 3 CN/H 2 O (3/4/3), and 1 μl sample was loaded onto the sample plate, air dried at room temperature, and then, 1 μl matrix solution (CHCA 4 mg/ml in CH 3 CN/H 2 O (1/1) with 0.1% TFA and doped with serine 20 mM) was added to the spot without mixing. The signal at m/z 1009.6 corresponds to HOCH 2 CO-Leu-Arg-His-Leu-Thr-Asp(Ala-NH 2 )-Ala-Gly-NH 2 …”

“…Figure 1 shows an example of a MALDI mass spectrum and MALDI tandem mass spectrum of peptides eluted from one bead of the combinatorial library. The signal at m/z 1009.6 corresponds HOCH 2 CO-LeuArg-His-Leu-Thr-Asp(Ala-NH 2 )-Ala-Gly-NH 2 Peptide sequences could be deduced from the tandem mass spectra (Figure 1 After library screening and MS sequencing, selected peptide ligands are resynthesised in larger amounts to study their performance for their intended use. In order to assure cyclic ligand stability, the Ala-glycolamidic ester bond can be replaced by an Ala-Gly amide bond by assembling Gly instead of glycolic acid.…”

Generation and Refinement of Peptide Mimetic Ligands for Paratope-Specific Purification of Monoclonal Antibodies

“…When this is translated to an affinity support (such as CNBractivated Sepharose) the orientation of the ligand is reversed and its ability to bind antibody is severely reduced. Other researchers have found that orienta- tion of small peptide ligands may be irrelevant to purification performance (31); therefore, the problem may be system specific. In our approach, the hydrophobic phenylalanine and leucine residues are critical in the MAb4155-peptide interaction, suggesting the requirement to be spaced away from the surface of the matrix.…”

“…The binding of a peptide to an antibody paratope is ultimately controlled by the complementarity of the surfaces (31). This includes charge-charge and hydrophobic interactions as well as three-dimensional conformation.…”

Stability Evaluation of Immobilized Peptides Towards Proteases by Mass Spectrometry

“…Short peptides are widely used as ligands in affinity chromatography purification of proteins [1,2]. However, peptidases and proteases present in the crude sample may degrade immobilized peptides, shortening the affinity support useful life.…”