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.

Biocatalysis is the activity to synthesize chemicals using biological entities, in particular enzymes. Natural enzymes often need to be optimized before being used in industrial processes, and directed evolution is an approach that is well suited for the creation of these improved industrial biocatalysts. Directed evolution involves creating a library of mutant genes and then sorting them based on the associated phenotypes. Main library creation and sorting protocols are compared here, with an emphasis on semirational approaches to library creation and fast screening or pool selection techniques. Representative reports of improved catalytic activity, enzyme stability, and specificity are discussed. Finally, an insight is given on recent technological developments to make enzymes more suitable for biocatalysis. These new technologies could favor the switch of numerous processes from chemistry to biocatalysis soon.

Transfer of vSAG7, the endogenous superantigen encoded in the Mtv7 locus, from MHC class II− to MHC class II+ cells has been suggested to occur both in vivo and in vitro. This transfer usually leads to the activation and deletion of T cells expressing responsive Vβs. However, there is no direct molecular evidence for such a transfer. We have developed an in vitro system which confirms this property of vSAGs. vSAG7 was transfected into a class II− murine fibroblastic line. Coculture of these cells with class II+ cells and murine T cell hybridomas expressing the specific Vβs led to high levels of IL-2 production which was specifically inhibited by vSAG7- and MHC class II–specific mAbs. Moreover, injection of vSAG7+ class II− cells in mice led to expansion of Vβ6+ CD4+ cells. We show that this transfer activity is paracrine but does not require cell-to-cell contact. Indeed, vSAG7 was transferred across semi-permeable membranes. Transfer can occur both from class II− and class II+ cells, indicating that MHC class II does not sequester vSAG7. Finally, competition experiments using bacterial toxins with well defined binding sites showed that the transferred vSAG7 fragment binds to the α1 domain of HLA-DR.