scholarly journals Design, Characterization, and Evaluation of scFvCD133/rGelonin: A CD133-Targeting Recombinant Immunotoxin for Use in Combination with Photochemical Internalization

2019 ◽  
Vol 9 (1) ◽  
pp. 68 ◽  
Author(s):  
Cathrine Elisabeth Olsen ◽  
Lawrence H. Cheung ◽  
Anette Weyergang ◽  
Kristian Berg ◽  
Daniel A. Vallera ◽  
...  
Keyword(s):  
Endosomal Escape ◽  
Intracellular Accumulation ◽  
Ribosome Inactivating Protein ◽  
Gene Encoding ◽  
Photochemical Internalization ◽  
Recombinant Immunotoxin ◽  
Fold Reduction ◽  
Free Translation ◽  
A Cell ◽  
Cytotoxic Responses

The objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133high and CD133low cell lines and a CD133neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133high cells compared to CD133low cells. No cytotoxic responses were detected in either CD133high or CD133low cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133high and CD133low cancer cells.

Preparation and Optimization of a Cell-free Translation System fromVicia sativaGerm Lacking Ribosome-inactivating Protein Activity

1992 ◽  
Vol 43 (5) ◽  
pp. 729-737 ◽  
Author(s):  
F. J. ARIAS ◽  
M. A. ROJO ◽  
J. M. FERRERAS ◽  
R. IGLESIAS ◽  
R. MUÑOZ ◽  
...  
Keyword(s):  
Translation System ◽  
Ribosome Inactivating Protein ◽  
Protein Activity ◽  
Free Translation ◽  
A Cell

scholarly journals A Cell-Free Translation and Proteoliposome Reconstitution System for Functional Analysis of Plant Solute Transporters

2007 ◽  
Vol 48 (12) ◽  
pp. 1815-1820 ◽  
Author(s):  
Akira Nozawa ◽  
Hideaki Nanamiya ◽  
Takuji Miyata ◽  
Nicole Linka ◽  
Yaeta Endo ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Free Translation ◽  
A Cell ◽  
Solute Transporters

Cloning of the gene encoding TROP-2, a cell-surface glycoprotein expressed by human carcinomas

1995 ◽  
Vol 62 (5) ◽  
pp. 610-618 ◽  
Author(s):  
Mara Fornaro ◽  
Roberta Dell' Arciprete ◽  
Manuela Stella ◽  
Cecilia Bucci ◽  
Michele Nutini ◽  
...  
Keyword(s):  
Cell Surface ◽  
Surface Glycoprotein ◽  
Gene Encoding ◽  
Cell Surface Glycoprotein ◽  
Human Carcinomas ◽  
A Cell

scholarly journals Activation of the Kexin from Schizosaccharomyces pombeRequires Internal Cleavage of Its Initially Cleaved Prosequence

1998 ◽  
Vol 18 (1) ◽  
pp. 400-408 ◽  
Author(s):  
Dale Powner ◽  
John Davey
Keyword(s):  
Cleavage Site ◽  
Secretory Pathway ◽  
Catalytic Domain ◽  
Activation Process ◽  
Processing Enzymes ◽  
Free Translation ◽  
A Cell ◽  
Internal Site ◽  
Primary Cleavage ◽  
Full Activation

ABSTRACT Members of the kexin family of processing enzymes are responsible for the cleavage of many proproteins during their transport through the secretory pathway. The enzymes themselves are made as inactive precursors, and we investigated the activation process by studying the maturation of Krp1, a kexin from the fission yeastSchizosaccharomyces pombe. Using a cell-free translation-translocation system prepared from Xenopuseggs, we found that Krp1 is made as a preproprotein that loses the presequence during translocation into the endoplasmic reticulum. The prosequence is also rapidly cleaved in a reaction that is autocatalytic and probably intramolecular and is inhibited by disruption of the P domain. Prosequence cleavage normally occurs at Arg-Tyr-Lys-Arg102↓ (primary cleavage site) but can occur at Lys-Arg82 (internal cleavage site) and/or Trp-Arg99 when the basic residues are removed from the primary site. Cleavage of the prosequence is necessary but not sufficient for activation, and Krp1 is initially unable to process substrates presented in trans. Full activation is achieved after further incubation in the extract and is coincident with the addition of O-linked sugars. O glycosylation is not, however, essential for activity, and the crucial event appears to be cleavage of the initially cleaved prosequence at the internal site. Our results are consistent with a model in which the cleaved prosequence remains noncovalently associated with the catalytic domain and acts as an autoinhibitor of the enzyme. Inhibition is then relieved by a second (internal) cleavage of the inhibitory prosequence. Further support for this model is provided by our finding that overexpression of a Krp1 prosequence lacking a cleavable internal site dramatically reduced the growth rate of otherwise wild-type S. pombecells, an effect that was not seen after overexpression of the normal, internally cleavable, prosequence or prosequences that lack the Lys-Arg102 residues.

scholarly journals The 3' untranslated region of satellite tobacco necrosis virus RNA stimulates translation in vitro.

1993 ◽  
Vol 13 (6) ◽  
pp. 3340-3349 ◽  
Author(s):  
X Danthinne ◽  
J Seurinck ◽  
F Meulewaeter ◽  
M Van Montagu ◽  
M Cornelissen
Keyword(s):  
Tobacco Necrosis Virus ◽  
Translation System ◽  
Coding Region ◽  
Necrosis Virus ◽  
Untranslated Sequence ◽  
Virus Rna ◽  
Free Translation ◽  
Order Of Magnitude ◽  
A Cell

The RNA of satellite tobacco necrosis virus (STNV) is a monocistronic messenger that lacks both a 5' cap structure and a 3' poly(A) tail. We show that in a cell-free translation system derived from wheat germ, STNV RNA lacking the 600-nucleotide trailer is translated an order of magnitude less efficiently than full-size RNA. Deletion analyses positioned the translational enhancer domain (TED) within a conserved hairpin structure immediately downstream from the coat protein cistron. TED enhances translation when fused to a heterologous mRNA, but the level of enhancement depends on the nature of the 5' untranslated sequence and is maximal in combination with the STNV leader. The STNV leader and TED have two regions of complementarity. One of the complementary regions in TED resembles picornavirus box A, which is involved in cap-independent translation but which is located upstream of the coding region.

scholarly journals Breaking in and busting out: cell-penetrating peptides and the endosomal escape problem

2017 ◽  
Vol 8 (3-4) ◽  
pp. 131-141 ◽  
Author(s):  
Julia C. LeCher ◽  
Scott J. Nowak ◽  
Jonathan L. McMurry
Keyword(s):  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Great Promise ◽  
Delivery Methods ◽  
Oligomeric Proteins ◽  
Cell Penetrating ◽  
History Of ◽  
A Cell ◽  
Escape Problem ◽  
Primary Focus

AbstractCell-penetrating peptides (CPPs) have long held great promise for the manipulation of living cells for therapeutic and research purposes. They allow a wide array of biomolecules from large, oligomeric proteins to nucleic acids and small molecules to rapidly and efficiently traverse cytoplasmic membranes. With few exceptions, if a molecule can be associated with a CPP, it can be delivered into a cell. However, a growing realization in the field is that CPP-cargo fusions largely remain trapped in endosomes and are eventually targeted for degradation or recycling rather than released into the cytoplasm or trafficked to a desired subcellular destination. This ‘endosomal escape problem’ has confounded efforts to develop CPP-based delivery methods for drugs, enzymes, plasmids, etc. This review provides a brief history of CPP research and discusses current issues in the field with a primary focus on the endosomal escape problem, for which several promising potential solutions have been developed. Are we on the verge of developing technologies to deliver therapeutics such as siRNA, CRISPR/Cas complexes and others that are currently failing because of an inability to get into cells, or are we just chasing after another promising but unworkable technology? We make the case for optimism.

scholarly journals Understanding Cell Interactions Using Modular Nanoparticle Libraries

2019 ◽  
Vol 72 (8) ◽  
pp. 595 ◽  
Author(s):  
Georgina K. Such ◽  
Angus P. R. Johnston
Keyword(s):  
Immune Responses ◽  
Small Molecules ◽  
Active Form ◽  
Reticuloendothelial System ◽  
Endosomal Escape ◽  
Biological Interactions ◽  
Nanoparticle Delivery ◽  
A Cell ◽  
Nanoparticle Structure ◽  
The Right

Nanoparticle delivery systems have significant potential to facilitate the delivery of novel therapeutics, such as proteins, DNA or small molecules. However, there are multiple biological barriers that need to be overcome to deliver the cargo in an active form. These challenges include evading clearance by the reticuloendothelial system, minimising adverse immune responses, targeting specific cells and tissues, and trafficking into the right compartment of the cell. In this account, we will discuss how nanoparticle structure can be tuned to optimise biological interactions and thus improve the ability of nanoparticles to overcome these barriers. The focus of this article will be on controlling cell targeting and trafficking within a cell, e.g. endosomal escape.

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