OPTIMAL SCREENING OF SURFACE-DISPLAYED POLYPEPTIDE LIBRARIES

Cell surface display of polypeptide libraries combined with flow cytom etric cell sorting presents remarkable potential for enhancement of pr otein-ligand recognition properties. To maximize the utility of this a pproach, screening and purification conditions must be optimized to ta ke full advantage of the quantitative feature of this technique. In pa rticular, discrimination of improved library mutants from an excess of wild-type polypeptides is dependent upon an effective screening metho dology. Fluorescence discrimination profiles for improved library muta nts were derived from a mathematical model of expected cell fluorescen ce intensities for polypeptide libraries screened with fluorescent lig and. Profiles for surface-displayed libraries under equilibrium or kin etic screening conditions demonstrate distinct discrimination optima f rom which optimal equilibrium and kinetic screening parameters were de rived. In addition, a statistical model of flow cytometrically analyze d cell populations indicates the importance of low-stringency sorting followed by amplification through regrowth and resorting at increased stringency. This analysis further yields quantitative recommendations for cell-sorting stringency.