Inntags: new tools for innocuous protein tagging

September 01, 2015

The study, published today at Nature Methods (the most prestigious journal for the presentation of results in methods development), proposes the use of two plant protein epitopes, named inntags, as the most innocuous and stable tagging tools in the study of physical and functional interactions of proteins.

Proteins and peptides of various sizes and shapes have been used since the early 80s to tag proteins with many different purposes, ranging from affinity purification to fluorescence-based microscopic detection in whole organisms. However, tagging strategies used nowadays run the risk that the native function of the protein may be abolished or compromised by interactions with the tag.

A study leaded by the Molecular Biology Institute of Barcelona and the Joint Programme for Computational Biology of the Institute for Research in Biomedicine (IRB Barcelona) and the Barcelona Supercomputing Center, and University of Barcelona, has analyzed the available list of polypeptides with known 3D structure to identify among them the most suitable for tagging purposes. Researches have selected the smallest protein domains that still display strong structural determinants to act as antigens, do not generate solubility issues, do not compromise cell fitness and cause no detectable functional and localization effects when fused to other target proteins.

A large series of bioinformatics tools were first used to scan through the entire planet proteome to select those proteins which could, in principle, have good tagging properties. After manual curation of the in silico results 12 tag candidates were tested in vivo, finding excellent or outstanding properties for all of them. Inntags maintain their integrity, stability, solubility in cell extracts, diffusional mobility and do not cause important functional perturbations that commonly used tags -such as MYC, FLAG or HA- do cause. Moreover, the tests have shown the applicability of Allergen Phl p2 and Heiven Isoform 2 in immunofluorescence and immunoprecipitation analysis of a series of proteins in mouse fibroblasts and hippocampal neurons.

As they are clearly more innocuous compared to commonly used tags, inntags may be the tools of choice to perform proteome-wide interactome studies, in situ analysis of proteins at the single-molecule level or when the target protein does not offer an obvious functional assay. The developed technology will open new possibilities for researchers in cellular biology, as they will provide them a neater, unbiased method to track proteins inside the cell.
-end-
Authors

The study has been led by Martí Aldea and Carme Gallego, Molecular Biology Institute of Barcelona (IBMB), CSIC, Barcelona, Catalonia, and Modesto Orozco, group leader at the IRB Barcelona, professor at the UB and director of Life Sciences departament at the BSC, Antibody BCN, Ltd, Immunostep Ltd, and Zagazig University have also contributed to this work.

Institute for Research in Biomedicine (IRB Barcelona)

Related Proteins Articles from Brightsurf:

New understanding of how proteins operate
A ground-breaking discovery by Centenary Institute scientists has provided new understanding as to the nature of proteins and how they exist and operate in the human body.

Finding a handle to bag the right proteins
A method that lights up tags attached to selected proteins can help to purify the proteins from a mixed protein pool.

Designing vaccines from artificial proteins
EPFL scientists have developed a new computational approach to create artificial proteins, which showed promising results in vivo as functional vaccines.

New method to monitor Alzheimer's proteins
IBS-CINAP research team has reported a new method to identify the aggregation state of amyloid beta (Aβ) proteins in solution.

Composing new proteins with artificial intelligence
Scientists have long studied how to improve proteins or design new ones.

Hero proteins are here to save other proteins
Researchers at the University of Tokyo have discovered a new group of proteins, remarkable for their unusual shape and abilities to protect against protein clumps associated with neurodegenerative diseases in lab experiments.

Designer proteins
David Baker, Professor of Biochemistry at the University of Washington to speak at the AAAS 2020 session, 'Synthetic Biology: Digital Design of Living Systems.' Prof.

Gone fishin' -- for proteins
Casting lines into human cells to snag proteins, a team of Montreal researchers has solved a 20-year-old mystery of cell biology.

Coupled proteins
Researchers from Heidelberg University and Sendai University in Japan used new biotechnological methods to study how human cells react to and further process external signals.

Understanding the power of honey through its proteins
Honey is a culinary staple that can be found in kitchens around the world.

Read More: Proteins News and Proteins Current Events
Brightsurf.com is a participant in the Amazon Services LLC Associates Program, an affiliate advertising program designed to provide a means for sites to earn advertising fees by advertising and linking to Amazon.com.