An affinity resin containing the peptide ligand Phe-Leu-Leu-Val-Pro-Leu (FLLVPL) has been developed for the purification of fibrinogen. The ligand was identified by screening a solid-phase combinatorial peptide library using an immunostaining technique. The specific binding of fibrinogen to the ligand has been characterized by isothermal calorimetry and adsorption isotherms and is dominated by both hydrophobic interactions and ionic interactions with the N-terminal free amino group. The effective association constant of fibrinogen was substantially higher when the peptide was immobilized on the resin than in solution; moreover, it increased with increasing peptide density, suggesting a cooperative binding effect. A low ionic strength buffer at pH 4 was used successfully to elute adsorbed fibrinogen from the column with high purity, retention of factor XIII crosslinking activity, and minimal, if any, loss of biological function. This general approach to ligand selection and characterization can be used to develop peptide ligands for the affinity purification of diverse proteins on a large scale.

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SummaryThe surfactant concentration is an important parameter for optimizing protein extraction with microemulsions. Equilibrium and kinetic experiments show that the surfactant concentration can be reduced by a factor of 20 compared to published data. The masstransfer coefficient is found to be only about 18% lower for small AOT concentrations.

SummaryThe kinetics of transfer of chymotrypsin from an aqueous into a reverse micellar phase and vice versa has been examined for a two-phase system with planar interface equipped with vibrating disks for separate mixing of both phases. The temperature dependence of the mass-transfer coefficients gives two values for the forward transfer (30 and 90 kJ/mol) depending on the temperature range. Only one value is obtained for the backward transfer (60kJ/mol).

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