Expression of random peptide fused to invasin on bacterial cell surface for selection of cell-targeting peptides

The protein invasin expressed on the cell surface of the pathogenic bacteri a Yersinia pseudotuberculosis mediates the entry of this bacterium into cul tured mammalian cells. We have developed a system for expression of random peptides on the cell surface of Escherichia coli (E. coli) by creation of a fusion hybrid between a peptide and the invasin protein. The fusion protei n constructs consist of part of the outer membrane domain of the invasin pr otein, six proline spacers, and a decamer of random peptides flanked by cys teine residues (CX10C). Peptides were constitutively expressed on the cell surface in the resulting random decamer peptide library, which we designate d as ESPEL ((E) under bar. coli (S) under bar urface (P) under bar eptide ( E) under bar xpression (L) under bar ibrary). The ESPEL was systematically screened for its binding affinity toward human cultured cells. Several bact erial clones were identified whose binding to human cells was mediated by p eptides expressed on the bacterial cell surface. Flow cytometric analysis s howed that both the identified bacterial clones and these corresponding che mically synthesized peptides bound to human cells specifically. The techniq ues described provide a new method that uses E. coli random peptide library to select targeting peptides for mammalian cells without any knowledge of the human cellular receptors. (C) 2000 Elsevier Science B.V. All rights res erved.