The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides |
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Authors: | Christmann, Andreas Walter, Kerstin Wentzel, Alexander Kratzner, Ralph Kolmar, Harald |
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Affiliation: | Abteilung für Molekulare Genetik und Präparative Molekularbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität, Grisebachstrasse 8, D-37077 Göttingen, Germany 1 These authors contributed equally to this work |
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Abstract: | The Ecballium elaterium trypsin inhibitor II (EETI-II), a memberof the squash family of protease inhibitors, is composed of28 amino acid residues and is a potent inhibitor of trypsin.Its compact structure is defined by a triple-stranded antiparallelß-sheet, which is held together by three intramoleculardisulfide bonds forming a cystine knot. In order to explorethe potential of the EETI-II peptide to serve as a structuralscaffold for the presentation of randomized oligopeptides, weconstructed two EETI-II derivatives, where the six-residue inhibitorloop was replaced by a 13-residue epitope of Sendai virus L-proteinand by a 17-residue epitope from human bone Gla-protein. EETI-IIand derived variants were produced via fusion to maltose bindingprotein MalE. By secretion of the fusion into the periplasmicspace, fully oxidized and correctly folded EETI-II was obtainedin high yield. EETI-II and derived variants could be presentedon the Escherichia coli outer membrane by fusion to truncatedLpp'OmpA', which comprises the first nine residues ofmature lipoprotein plus the membrane spanning ß-strandfrom residues 4666 of OmpA protein. Gene expression wasunder control of the strong and tightly regulated tetA promoter/operator.Cell viability was found to be drastically reduced by high levelexpression of Lpp'OmpA'EETI-II fusion protein.To restore cell viability, net accumulation of fusion proteinin the outer membrane was reduced to a tolerable level by introductionof an amber codon at position 9 of the lpp' sequence and utilizingan amber suppressor strain as expression host. Cells expressingEETI-II variants containing an epitope were shown to be surfacelabeled with the respective monoclonal antibody by indirectimmunofluorescence corroborating the cell surface exposure ofthe epitope sequences embedded in the EETI-II cystine knot scaffold.Cells displaying a particular epitope sequence could be enriched107-fold by combining magnetic cell sorting with fluorescence-activatedcell sorting. These results demonstrate that E.coli cell surfacedisplay of conformationally constrained peptides tethered tothe EETI-II cystine knot scaffold has the potential to becomean effective technique for the rapid isolation of small peptidemolecules from combinatorial libraries that bind with high affinityto acceptor molecules. |
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Keywords: | combinatorial libraries/ cystine knot/ EETI-II/ FACS/ squash inhibitor/ surface display |
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