Expression and cytosolic assembly of the S-layer fusion protein mSbsC-EGFP in eukaryotic cells

BACKGROUND: Native as well as recombinant bacterial cell surface layer (S-layer) protein of Geobacillus (G.) stearothermophilus ATCC 12980 assembles to supramolecular structures with an oblique symmetry. Upon expression in E. coli, S-layer self assembly products are formed in the cytosol. We tested the expression and assembly of a fusion protein, consisting of the mature part (aa 31–1099) of the S-layer protein and EGFP (enhanced green fluorescent protein), in eukaryotic host cells, the yeast Saccharomyces cerevisiae and human HeLa cells. RESULTS: Upon expression in E. coli the recombinant mSbsC-EGFP fusion protein was recovered from the insoluble fraction. After denaturation by Guanidine (Gua)-HCl treatment and subsequent dialysis the fusion protein assembled in solution and yielded green fluorescent cylindric structures with regular symmetry comparable to that of the authentic SbsC. For expression in the eukaryotic host Saccharomyces (S.) cerevisiae mSbsC-EGFP was cloned in a multi-copy expression vector bearing the strong constitutive GPD1 (glyceraldehyde-3-phosophate-dehydrogenase) promoter. The respective yeast transfomants were only slightly impaired in growth and exhibited a needle-like green fluorescent pattern. Transmission electron microscopy (TEM) studies revealed the presence of closely packed cylindrical structures in the cytosol with regular symmetry comparable to those obtained after in vitro recrystallization. Similar structures are observed in HeLa cells expressing mSbsC-EGFP from the Cytomegalovirus (CMV IE) promoter. CONCLUSION: The mSbsC-EGFP fusion protein is stably expressed both in the yeast, Saccharomyces cerevisiae, and in HeLa cells. Recombinant mSbsC-EGFP combines properties of both fusion partners: it assembles both in vitro and in vivo to cylindrical structures that show an intensive green fluorescence. Fusion of proteins to S-layer proteins may be a useful tool for high level expression in yeast and HeLa cells of otherwise instable proteins in their native conformation. In addition the self assembly properties of the fusion proteins allow their simple purification. Moreover the binding properties of the S-layer part can be used to immobilize the fusion proteins to various surfaces. Arrays of highly ordered and densely structured proteins either immobilized on surfaces or within living cells may be advantageous over the respective soluble variants with respect to stability and their potential interference with cellular metabolism

Synthesis and Characterization of an Epidermal Growth Factor Receptor selective Ru(II) Polypyridyl‐Nanobody Conjugate as a Photosensitizer for Photodynamic Therapy

International audienceThere is currently a surge for the development of novel photosensitizers (PSs) for photodynamic therapy (PDT) since those currently approved are not completely ideal. Among the tested compounds, we have previously investigated the use of Ru(II) polypyridyl complexes with a [Ru(bipy)2(dppz)]2+ and [Ru(phen)2(dppz)]2+ scaffold (bipy = 2,2'‐bipyridine; dppz = dipyrido[3,2‐a:2′,3′‐c]‐phenazine, phen = 1,10‐phenanthroline). These complexes selectively target DNA. However, since DNA is ubiquitous, it would be of great interest to increase the selectivity of our PDT PSs by linking them to a targeting vector in view of targeted PDT. Herein, we present the synthesis, characterization and in‐depth photophysical evaluation of a nanobody‐containing Ru(II) polypyridyl conjugate selective for the epidermal growth factor receptor (EGFR) in view of targeted PDT. Using ICP‐MS and confocal microscopy, we could demonstrate that our conjugate had a high selectivity for the EGFR receptor, which is a crucial oncological target as it is overexpressed and/or deregulated in a variety of solid tumors. However, contrary to expectations, this conjugate was found to not produce reactive oxygen species (ROS) in cancer cells and to be therefore not phototoxic

Tetranuclear Cu(II)-chiral complexes: synthesis, characterization and biological activity

Tetranuclear chiral Cu(ii)-Schiff-base complexes S-1 and R-1, were synthesised using enantiomerically pure (S)-(H(2)vanPheol) and (R)-(H(2)vanPheol) ligands respectively in the ratio of 1 : 1 of Cu(NO(3))(2) to (S/R)-(H(2)vanPheol) in MeOH at room temperature. A pair of polynuclear chiral Cu(ii)-cluster complexes were characterized using single-crystal X-ray diffraction, elemental analysis, infrared and CD spectroscopy. The results revealed the importance of these chiral ligands encouraging the arrangement of copper metal in non-centrosymmetric polar packing. The potential of the novel [Cu(4)(S/R-vanPheol)(2)(S/R-HvanPheol)(2)(CH(3)OH)(2)](NO(3))(2) complexes as biologically active compounds was assessed in particular regarding their anti-proliferative and anti-microbial properties

The s-layer glycome-adding to the sugar coat of bacteria

This work was supported by the Austrian Science Fund FWF, projects P19047-B12, P20605-B12, P21954-B20 (to C. Sch¨affer), and P20745-B11 (to P. Messner). Zarschler and Ristl were supported by the Hochschuljubil¨aumsstiftung der Stadt Wien, Projects H-2229-2007 (to K. Zarschler) and H-1897-2008 (to R. Ristl).The amazing repertoire of glycoconjugates present on bacterial cell surfaces includes lipopolysaccharides, capsular polysaccharides, lipooligosaccharides, exopolysaccharides, and glycoproteins. While the former are constituents of Gram-negative cells, we review here the cell surface S-layer glycoproteins of Gram-positive bacteria. S-layer glycoproteins have the unique feature of self-assembling into 2D lattices providing a display matrix for glycans with periodicity at the nanometer scale. Typically, bacterial S-layer glycans are O-glycosidically linked to serine, threonine, or tyrosine residues, and they rely on a much wider variety of constituents, glycosidic linkage types, and structures than their eukaryotic counterparts. As the S-layer glycome of several bacteria is unravelling, a picture of how S-layer glycoproteins are biosynthesized is evolving. X-ray crystallography experiments allowed first insights into the catalysis mechanism of selected enzymes. In the future, it will be exciting to fully exploit the S-layer glycome for glycoengineering purposes and to link it to the bacterial interactome.Publisher PDFPeer reviewe

New insights into the pretargeting approach to image and treat tumours

Tumour pretargeting is a promising strategy for cancer diagnosis and therapy allowing for the rational use of long circulating, highly specific monoclonal antibodies (mAbs) for both non-invasive cancer radioimmunodetection (RID) and radioimmunotherapy (RIT). In contrast to conventional RID/RIT where the radionuclides and oncotropic vector molecules are delivered as presynthesised radioimmunoconjugates, the pretargeting approach is a multistep procedure that temporarily separates targeting of certain tumour-associated antigens from delivery of diagnostic or therapeutic radionuclides. In principle, unlabelled, highly tumour antigen specific mAb conjugates are, in a first step, administered into a patient. After injection, sufficient time is allowed for blood circulation, accumulation at the tumour site and subsequent elimination of excess mAb conjugates from the body. The small fast-clearing radiolabelled effector molecules with a complementary functionality directed to the prelocalised mAb conjugates are then administered in a second step. Due to its fast pharmacokinetics, the small effector molecules reach the malignant tissue quickly and bind the local mAb conjugates. Thereby, corresponding radioimmunoconjugates are formed in vivo and, consequently, radiation doses are deposited mainly locally. This procedure results in a much higher tumour/non-tumour (T/NT) ratio and is favourable for cancer diagnosis and therapy as it substantially minimises the radiation damage to non-tumour cells of healthy tissues. The pretargeting approach utilises specific non-covalent interactions (e.g. strept(avidin)/biotin) or covalent bond formations (e.g. inverse electron demand Diels–Alder reaction) between the tumour bound antibody and radiolabelled small molecules. This tutorial review descriptively presents this complex strategy, addresses the historical as well as recent preclinical and clinical advances and discusses the advantages and disadvantages of different available variations

Towards Utilising Photocrosslinking of Polydiacetylenes for the Preparation of { extquotedblleft}Stealth{ extquotedblright} Upconverting Nanoparticles

We demonstrate a novel strategy for preparing hydrophilic upconverting nanoparticles (UCNPs) by harnessing the photocrosslinking ability of diacetylenes. Replacement of the hydrophobic oleate coating on the UCNPs with 10,12- pentacosadiynoic acid, followed by overcoating with diacetylene phospholipid and subsequent photocrosslinking under 254 nm irradiation produces water-dispersible polydiacetylene- coated UCNPs. These UCNPs resist the formation of a biomolecular corona and show great colloidal stability. Furthermore, amine groups on the diacetylene phospholipid allow for functionalisation of the UCNPs with, for example, radiolabels or targeting moieties. These results demonstrate that this new surface-coating method has great potential for use in the preparation of UCNPs with improved biocompatibility

Towards Utilising Photocrosslinking of Polydiacetylenes for the Preparation of “Stealth” Upconverting Nanoparticles

We demonstrate a novel strategy for preparing hydrophilic upconverting nanoparticles (UCNPs) by harnessing the photocrosslinking ability of diacetylenes. Replacement of the hydrophobic oleate coating on the UCNPs with 10,12- pentacosadiynoic acid, followed by overcoating with diacetylene phospholipid and subsequent photocrosslinking under 254 nm irradiation produces water-dispersible polydiacetylene- coated UCNPs. These UCNPs resist the formation of a biomolecular corona and show great colloidal stability. Furthermore, amine groups on the diacetylene phospholipid allow for functionalisation of the UCNPs with, for example, radiolabels or targeting moieties. These results demonstrate that this new surface-coating method has great potential for use in the preparation of UCNPs with improved biocompatibility

The s-layer glycome-adding to the sugar coat of bacteria

The amazing repertoire of glycoconjugates present on bacterial cell surfaces includes lipopolysaccharides, capsular polysaccharides, lipooligosaccharides, exopolysaccharides, and glycoproteins. While the former are constituents of Gram-negative cells, we review here the cell surface S-layer glycoproteins of Gram-positive bacteria. S-layer glycoproteins have the unique feature of self-assembling into 2D lattices providing a display matrix for glycans with periodicity at the nanometer scale. Typically, bacterial S-layer glycans are O-glycosidically linked to serine, threonine, or tyrosine residues, and they rely on a much wider variety of constituents, glycosidic linkage types, and structures than their eukaryotic counterparts. As the S-layer glycome of several bacteria is unravelling, a picture of how S-layer glycoproteins are biosynthesized is evolving. X-ray crystallography experiments allowed first insights into the catalysis mechanism of selected enzymes. In the future, it will be exciting to fully exploit the S-layer glycome for glycoengineering purposes and to link it to the bacterial interactome