Designed affinity ligands to capture human serum albumin.

Human serum albumin (HSA) in an important therapeutic agent and disease biomarker, with an increasing market demand. By proteins and drugs that bind to HSA as inspiration, a combinatorial library of 64 triazine-based ligands was rationally designed and screened for HSA binding at physiological conditions. Two triazine-based lead ligands (A3A2 and A6A5), presenting more than 50% HSA bound and high enrichment factors, were selected for further studies. Binding and elution conditions for HSA purification from human plasma were optimized for both ligands. The A6A5 adsorbent yielded a purified HSA sample with 98% purity at 100% recovery yield under mild binding and elution conditions.

Affitins for protein purification by affinity magnetic fishing.

Currently most economical and technological bottlenecks in protein production are placed in the downstream processes. With the aim of increasing the efficiency and reducing the associated costs, various affinity ligands have been developed. Affitins are small, yet robust and easy to produce, proteins derived from the archaeal extremophilic "7kDa DNA-binding" protein family. By means of combinatorial protein engineering and ribosome display selection techniques, Affitins have shown to bind a diversity of targets. In this work, two previously developed Affitins (anti-lysozyme and anti-IgG) were immobilized onto magnetic particles to assess their potential for protein purification by magnetic fishing. The optimal lysozyme and human IgG binding conditions yielded 58mg lysozyme/g support and 165mgIgG/g support, respectively. The recovery of proteins was possible in high yield (≥95%) and with high purity, namely ≥95% and 81%, when recovering lysozyme from Escherichia coli supernatant and IgG from human plasma, respectively. Static binding studies indicated affinity constants of 5.0×10(4)M(-1) and 9.3×10(5)M(-1) for the anti-lysozyme and anti-IgG magnetic supports. This work demonstrated that Affitins, which can be virtually evolved for any protein of interest, can be coupled onto magnetic particles creating novel affinity adsorbents for purification by magnetic fishing.