The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides

The Ecballium elaterium trypsin inhibitor II (EETI-II), a member of the squ ash family of protease inhibitors, is composed of 28 amino acid residues an d is a potent inhibitor of trypsin. Its compact structure is defined by a t riple-stranded antiparallel beta-sheet, which is held together by three int ramolecular disulfide bonds forming a cystine knot. In order to explore the potential of the EETI-II peptide to serve as a structural scaffold for the presentation of randomized oligopeptides, we constructed two EETI-II deriv atives, where the six-residue inhibitor loop was replaced by a 13-residue e pitope of Sendai virus L-protein and by a 17-residue epitope from human bon e Gla-protein, EETI-II and derived variants were produced via fusion to mal tose binding protein MalE. By secretion of the fusion into the periplasmic space, fully oxidized and correctly folded EETI-II was obtained in high yie ld, EETI-II and derived variants could be presented on the Escherichia coli outer membrane by fusion to truncated Lpp'-OmpA: which comprises the first nine residues of mature lipoprotein plus the membrane spanning beta-strand from residues 46-66 of OmpA protein. Gene expression was under control of the strong and tightly regulated tetA promoter/operator. Cell viability was found to be drastically reduced by high level expression of Lpp'-OmpA'-EET I-II fusion protein, To restore cell viability, net accumulation of fusion protein in the outer membrane was reduced to a tolerable level by introduct ion of an amber codon at position 9 of the lpp' sequence and utilizing an a mber suppressor strain as expression host. Cells expressing EETI-II variant s containing an epitope were shown to be surface labeled with the respectiv e monoclonal antibody by indirect immunofluorescence corroborating the cell surface exposure of the epitope sequences embedded in the EETI-II cystine knot scaffold. Cells displaying a particular epitope sequence could be enri ched 10(7)-fold by combining magnetic cell sorting with fluorescence-activa ted cell sorting, These results demonstrate that E,coli cell surface displa y of conformationally constrained peptides tethered to the EETI-II cystine knot scaffold has the potential to become an effective technique for the ra pid isolation of small peptide molecules from combinatorial libraries that bind with high affinity to acceptor molecules.