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

Abstract

The Ecballium elaterium trypsin inhibitor II (EETI-II), a member of the squash family of protease inhibitors, is composed of 28 amino acid residues and is a potent inhibitor of trypsin. Its compact structure is defined by a triple-stranded antiparallel beta-sheet, which is held together by three intramolecular 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 derivatives, where the six-residue inhibitor loop was replaced by a 13-residue epitope of Sendai virus L-protein and by a 17-residue epitope from human bone Gla-protein. EETI-II and derived variants were produced via fusion to maltose binding protein MalE. By secretion of the fusion into the periplasmic space, fully oxidized and correctly folded EETI-II was obtained in high yield. 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'-EETI-II fusion protein. To restore cell viability, net accumulation of fusion protein in the outer membrane was reduced to a tolerable level by introduction of an amber codon at position 9 of the lpp' sequence and utilizing an amber suppressor strain as expression host. Cells expressing EETI-II variants containing an epitope were shown to be surface labeled with the respective 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 enriched 10(7)-fold by combining magnetic cell sorting with fluorescence-activated cell sorting. These results demonstrate that E.coli cell surface display of conformationally constrained peptides tethered to the EETI-II cystine knot scaffold has the potential to become an effective technique for the rapid isolation of small peptide molecules from combinatorial libraries that bind with high affinity to acceptor molecules.

Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation

“…The culture was grown at 15°C, and the expression of the Bla variant, which is under T7 promoter control, was induced at an OD 600 of 0.6 by the addition of 1 mM IPTG. The ␤-lactamase variant proteins were purified by metal chelate affinity chromatography (9).…”

Display of Passenger Proteins on the Surface ofEscherichia coliK-12 by the EnterohemorrhagicE. coliIntimin EaeA

“…The resulting PCR product was digested with XbaI and MunI and ligated to XbaI/EcoRI-digested vector pASKC21-EETI to give pASKInt1. pASKC21-EETI is a derivative of plasmid pASK21-EETI (9), where the coding sequence for an E epitope was placed 5Ј to the Ecballium elaterium trypsin inhibitor II (EETI-II) coding sequence. It contains a chloramphenicol resistance marker instead of the ampicillin resistance gene which has codons 8 to 281 deleted.…”

“…The interleukin 4 (IL-4)-encoding gene was amplified by PCR using the vector pRPR9IL-4FD (29) as a template and the primer pair IL-4-up and IL-4send-lo. IL-4send-lo hybridizes at the 3Ј end of the IL-4 gene and introduces the nucleotide sequence encoding the 13-residue epitope from the Sendai virus L protein (9). The resulting PCR product was digested with SmaI and BamHI and ligated to similarly digested pASKInt100.…”

“…The MalE-RGD fusion protein was affinity purified (34) using an amylose column (Pharmacia). MalE-EETI-CK RGD , which contains six carboxy-terminal histidines, was purified by metal chelate affinity chromatography as described elsewhere (9). The protein concentration was determined from the A 280 (38).…”

“…Short peptides (less than approximately 50 amino acids [aa]) were successfully displayed on the cell surface by insertion into surface-exposed loops of fimbrial proteins (24) or outer membrane proteins like LamB (7) or PhoE (2). Larger passenger domains could be presented on the E. coli cell surface by insertion into a surface-exposed domain of the E. coli flagellin FliC (50), by carboxy-terminal fusion to Lpp-OmpA (a hybrid protein consisting of parts of the E. coli lipoprotein Lpp and OmpA protein [9,12]), by using the peptidoglycan-associated lipoprotein PAL as an outer membrane anchor (15), by amino-terminal fusion to the ␤-domain of immunoglobulin A (IgA) protease of Neisseria gonorrhoeae and other autotransporters (22,30,35), or by C-terminal fusion to InaZ, the ice nucleation protein of Pseudomonas syringae (18).Pathogenic gram-negative bacteria have developed several distinct secretion mechanisms for the efficient surface display of binding domains, which specifically interact with their complementary receptors on host cell surfaces (23). Among them, intimins and invasins are members of a family of bacterial adhesins which specifically interact with diverse eukaryotic cell surface receptors, thereby mediating bacterial adherence and invasion (48).…”