Selection of bacterial inner-membrane anchor polypeptides

Abstract

The invention overcomes the deficiencies of the prior art by providing a rapid approach for isolating polypeptides capable of anchoring heterologous polypeptides to a bacterial inner membrane. In the technique, libraries of candidate anchor polypeptides are expressed as fusions with a heterologous polypeptide that is capable of being detected when bound to the inner membrane. In bacteria expressing a functional anchor sequence, the heterologous polypeptide becomes bound to outer face of the inner membrane. Bacteria with the functional anchor sequence can be identified by removing the outer membrane to remove non-anchored heterologous polypeptide followed by detection of anchored heterologous polypeptide. Such bacteria may be detected in numerous ways, including use of direct fluorescence or secondary antibodies that are fluorescently labeled, allowing use of efficient techniques such as fluorescence activated cell sorting (FACS).