Functional mapping of the A2 domain from human factor VIII.

Coagulation factor VIII (FVIII) is a multidomain glycoprotein in which the FVIII A2 domain is a key structural element. We aimed at identifying residues within FVIII A2 domain that are crucial for the maintenance of the cofactor function. A high number (n=206) of mutants were generated by substituting original residues with alanine.

Functional mapping of factor VIII C2 domain.

The factor VIII (FVIII) is a cofactor of the coagulation cascade. The FVIII C2 domain is a critical domain that participates in the interactions with the von Willebrand factor and the phospholipidic surfaces. To assess the importance of each residue of this domain in the maintenance of the structure and the function of FVIII, a number (n=139) of mutants were generated by substituting the original residues, from Ser2173 to Gly2325, by an alanine.

An activity-independent selection system of thermostable protein variants.

We describe an activity-independent method for the selection of thermostable mutants of any protein. It is based on a fusion construct comprising the protein of interest and a thermostable antibiotic resistance reporter, in such a way that thermostable mutants provide increased resistance in a thermophile. We isolated thermostable mutants of three human interferons and of two enzymes to demonstrate the applicability of the system.

High-throughput site-directed mutagenesis using oligonucleotides synthesized on DNA chips.

Site-directed mutagenesis has greatly helped researchers both to understand the precise role of specific residues in coding sequences and to generate variants of proteins that have acquired new characteristics. Today's demands for more complete functional cartographies of proteins and advances in selection and screening technologies require that site-directed mutagenesis be adapted for high-throughput applications. We describe here the first generation of a library of single and multiple site-directed mutants using a mixture of oligonucleotides synthesized on DNA chips.