ABSTRACT
Development of agents for theranostics implies combining the targeting module, the effector module, and the detection module within the same complex or recombinant protein. We have constructed, isolated, and characterized the 4D5scFv-mCherry-PE(40) protein, which exhibits fluorescent properties and specifically binds to cancer cells expressing the HER2 receptor and reduces their viability. The ability of the obtained targeted antitumor agent 4D5scFv-mCherry-PE(40) to selectively stain the HER2-positive cells and its highly selective cytotoxicity against these cells make the obtained targeted recombinant protein 4D5scFv-mCherry-PE(40) a promising theranostic agent for the diagnostics and therapy of HER2-positive human tumors.
Subject(s)
Immunotoxins/pharmacology , Luminescent Proteins/pharmacology , Single-Chain Antibodies/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/isolation & purification , Antineoplastic Agents/pharmacology , Antineoplastic Agents/toxicity , CHO Cells , Cell Line, Tumor , Cell Survival/drug effects , Chromatography, Affinity , Cricetulus , Escherichia coli , Fluorescence , Genetic Vectors , Humans , Immunotoxins/isolation & purification , Immunotoxins/toxicity , Luminescent Proteins/chemical synthesis , Luminescent Proteins/isolation & purification , Luminescent Proteins/toxicity , Microscopy, Fluorescence , Receptor, ErbB-2/metabolism , Recombinant Proteins/chemical synthesis , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Recombinant Proteins/toxicity , Single-Chain Antibodies/isolation & purification , Single-Chain Antibodies/toxicityABSTRACT
Multicolour labelling with fluorescent proteins is frequently used to differentially highlight specific structures in living systems. Labelling with fusion proteins is particularly demanding and is still problematic with the currently available palette of fluorescent proteins that emit in the red range due to unsuitable subcellular localization, protein-induced toxicity and low levels of labelling efficiency. Here we report a new monomeric red fluorescent protein, called FusionRed, which demonstrates both high efficiency in fusions and low toxicity in living cells and tissues.
Subject(s)
Luminescent Proteins/toxicity , Animals , Cell Death/drug effects , Chromatography, High Pressure Liquid , HeLa Cells , Humans , Microscopy, Fluorescence , Models, Molecular , Protein Multimerization/drug effects , Recombinant Fusion Proteins/toxicity , Xenopus laevis , Red Fluorescent ProteinABSTRACT
Using the yeast two-hybrid (YTH) system we have uncovered interaction of the hRPB11cα minor isoform of Homo sapiens RNA polymerase II hRPB11 (POLR2J) subunit with three different subunits of the human translation initiation factor eIF3 (hEIF3): eIF3a, eIF3i, and eIF3m. One variant of eIF3m identified in the study is the product of translation of alternatively spliced mRNA. We have named a novel isoform of this subunit eIF3mß. By means of the YTH system we also have shown that the new eIF3mß isoform interacts with the eIF3a subunit. Whereas previously described subunit eIF3mα (GA17) has clear cytoplasmic localization, the novel eIF3mß isoform is detected predominantly in the cell nucleus. The discovered interactions of the hRPB11cα isoform with several hEIF3 subunits demonstrate a new type coordination between transcription and the following (downstream) stages of gene expression (such as mRNA transport from nucleus to the active ribosomes in cytoplasm) in Homo sapiens and point out the possibility of existence of nuclear hEIF3 subcomplexes.
Subject(s)
Eukaryotic Initiation Factor-3/metabolism , RNA Polymerase II/metabolism , Eukaryotic Initiation Factor-3/genetics , Humans , Protein Binding , RNA Polymerase II/genetics , Two-Hybrid System TechniquesABSTRACT
We briefly review the current literature where optogenetics has been used to study various aspects of astrocyte physiology in vitro and in vivo. This includes both genetically engineered Ca(2+) sensors and effector proteins, such as channelrhodopsin. We demonstrate how the ability to target astrocytes with cell-specific viral vectors to express optogenetic constructs helped to unravel some previously unsuspected roles of these inconspicuous cells.
Subject(s)
Astrocytes/physiology , Genetic Engineering/methods , Optics and Photonics/methods , Rhodopsin/physiology , Animals , Astrocytes/metabolism , Calcium/metabolism , Light , Nerve Net/physiologyABSTRACT
Green fluorescent protein from Aequorea victoria and its many homologs are now widely used in basic and applied research. These genetically encoded fluorescent markers can detect localization of cell proteins and organelles in living cells and also cells and tissues in living organisms. Unique instruments and methods for studies of molecular biology of a cell and high throughput drug screenings are based on fluorescent proteins. This review deals with the most intensively evolving directions in this field, the development of genetically encoded sensors. Changes in their spectral properties are used for monitoring of cell enzyme activities or changes in concentrations of particular molecules.
