Designed monomeric streptavidin for labeling cells and dependent biotinylation proximity

Apr 1, 2016 | | Say something

Designed monomeric streptavidin for labeling cells and dependent biotinylation proximity ;

(A) tetramer streptavidin (left) is designed to form a monomer binds biotin with low affinity nM (right). (B) HEK293 cells showing biotinylated PPC (above) were fluorescently labeled with Alexa 488 conjugated MSA (bottom). Fluorescent images taken in i) t = 0 and ii) 50 min indicate little dissociation of bound MSA. (C) mSA domain fused to Z was used to form a noncovalent complex with the antibody and biotin reactive recruit a protein of interest, Erk-2. Biotinylated proteins were affinity purified using immobilized streptavidin and analyzed (for example, SDS-PAGE, Western blotting, mass spectrometry) to identify all the proteins that interact with Erk-2. Credit: TECHNOLOGY

A team of researchers from the University of Buffalo in Amherst, New York have successfully designed a new variant of streptavidin which forms a stable monomer and is able to detect monovalent biotin. Streptavidin is widely used for the detection of biotinylated ligands, but can cause crosslinking objective may interfere with the observations on a molecular level. The streptavidin monomer engineering (MSA) binds biotinylated targets without crosslinking, since it has a single site binding biotin to. As structural monomer, MSA can also be genetically fused to another protein to form a biotin-binding tag. Binding of a ligand biotinylated mSA domain fused is useful in many biochemical and cell biological studies. MSA optimization design and its possible applications in labeling studies and protein-protein interactions are described in the next 2016 magazine TECHNOLOGY .

“The native interaction streptavidin-biotin has limitations arising from the forced tetrameric structure streptavidin. A small protein domain, only binds biotin as MSA, extends the usefulness of the system streptavidin-biotin, allowing monovalent biotin detection and creation of gene fusions, “says professor Sheldon Park, Ph.D., of the University of Buffalo and principal investigator on the paper.

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and its homologs streptavidin all oligomers form, so it is difficult to use them as gene fusions. The group previously reported engineering a stable monomeric protein that binds with low affinity nM biotin and shown that molecule may be fused to GFP to create a bifunctional molecule. In this study, the group optimized design and demonstrated how engineering the molecule can be used in biotechnology. A significant improvement in the binding characteristics of mSA includes stabilization biotin complex together to achieve consistent labeling of biotinylated targets in time. For example, using mSA conjugated to a fluorophore, receptors biotin on the cell surface can be stably, and in particular with the label for a period of 1 hour with little loss of signal, which should be useful in studies fluorescence microscopy.

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Unlike oligomeric streptavidin, MSA can easily be fused to a protein of interest (POI) to construct a recombinant label for binding biotin. The current study explores the use of fusion mSA in the vicinity of-pending biotinylation-a technique that has proven useful in proteomics research. dependent biotinylation proximity enzymatic or chemical methods used to selectively biotinylated molecules interacting with POI, so that they can be affinity purified using immobilized streptavidin. The authors showed that the MSA label can be used to recruit a kind of biotin and achieve specific biotinylation reagent molecules that interact directly or indirectly. Since only the co-location is required for this to work mSA, direct melting of MSA is not necessary. Instead, MSA, could be recruited to the PDI by a number of noncovalent interactions, for example with an antibody in order to target molecules interacting for biotinylation. The design provides a high level of modularity and works only transiently interacting molecules, such as an enzyme and its substrate.

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UB team is working to further reduce biotin dissociation rate in order to optimize the efficiency of even more marked. To this end, the crystal structure of MSA (PDB 4JNJ) is used to model the effects of various mutations before they are tested in the laboratory. The improved biochemical characteristics and size mSA significantly lower (25% roughtly streptavidin) create new opportunities in biotechnology by leveraging existing infrastructure and the ubiquity of streptavidin-biotin system to study new interactions.

This article was originally published on medicalxpress, Read the original article

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