Directed evolution towards protease-resistant hirudin variants.

摘要: Abstract Hirudin, a thrombin-specific inhibitor, is efficiently digested and inactivated by proteases with pepsin- chymotrypsin-like specificity. Using combination of phage display selection high-throughput screening methods, several variants recombinant hirudin were generated. Only very few comprising amino acid substitutions in the amino-terminal domain (residues 1–5) carboxyl-terminal tail 49, 50, and/or 56, 57, 62–64) identified that showed thrombin inhibition activities similar to those wild-type polypeptide. Analysis protease susceptibility, however, revealed mutations, which conferred resistance, simultaneously diminish activity. This particularly apparent for region residues 56–64, forms large number electrostatic hydrophobic interactions crystal structure complex. Unlike hirudin, variant Pro 50 –⋯–His 56 –Asp 57 –⋯–Pro 62 –Pro 63 –His 64 completely resistant pepsin chymotrypsin cleavage; this at expense activity where there 100-fold increase IC50 value. The frequent replacement acids proline major cleavage sites indicates least enzymes may exhibit (conformational) specificity concerning P1 P2 positions. On basis these results, appear be general strategy design polypeptides are not susceptible digestion broader range different proteases.