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 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.     

Conversion of 5′-Methylthioadenosine into S-adenosylmethionine by yeast cells

5′-Methylthio[U-¹⁴C]adenosine was used as a culture supplement for Candida utilitis. The resulting S-adenosylmethionine was hydrolyzed into its structural components. Virtually none of the label of the pentose was found in the carbohydrate part of the intracellular S-adenosylmethionine. Much of it was present in the four-carbon chain of the methionine part of the sulfonium compound. The U-¹⁴C)-labeled adenine of 5′-methylthio[U-¹⁴C]adenosine did not contribute to the labeling of the amino acid component of the sulfonium compound.     

Spider mites of sugarcane in Australia: a review of grass-feeding Oligonychus Berlese (Acari: Prostigmata: Tetranychidae)

Abstract In many areas of the world, spider mites are significant pests of sugarcane. Australia is currently fortunate in lacking the most destructive species, and usually suffers only sporadic damage. Herein, we provide a key to the genera of spider mites associated with sugarcane, review the most significant genus, Oligonychus Berlese, and provide a key to the species of grass-feeding Oligonychus in the Australasian region. The species O. araneum Davis, O. digitatus Davis, O. grypus Baker and Pritchard, O. orthius Rimando, and O. oryzae (Hirst) are redescribed, while the Australian O. zanclopes sp. n. Beard and Walter from sugarcane and rice, O. turbelli sp. n. Beard and Walter, O. ephamnus sp. n. Beard and Walter and O. festucolus sp. n. Beard and Walter from other grasses, are newly described. Previous records of O. grypus in Australia appear to be misidentifications of what is described here as the new species O. zanclopes .