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Proximity Labeling: A Powerful Tool for Protein Complex Purification and Proteomic Mapping

Posted by Alyssa Cecchetelli on December 02, 2019

There are approximately 2-4 million proteins per cubic micron in bacteria, yeast, and mammalian cells (Milo, 2013). The number of interactions between these proteins is hard to imagine yet alone study.

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Topics: Plasmid Tags, Plasmids

Inntags: Innovative Protein Epitope Tagging

Posted by Mary Gearing on November 10, 2015

First described in the 1980s, protein tags are now one of the most useful items in a scientist’s toolbox. As we’ve covered in Plasmids 101, tags can help you determine localization of a protein of interest, purify it, or determine its expression level without the need for a custom antibody. There is one major caveat - a tag may interfere with protein localization and/or function, so each tagged protein must be tested carefully to ensure it retains the attributes of the native protein. Since this process takes a lot of time and energy, Martí Aldea and collaborators have created a set of “innocuous tags” (inntags) less likely to alter a protein’s properties.

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Topics: Plasmid Tags, Plasmids

Easing the Protein Purification Process with pCri

Posted by Mary Gearing on June 19, 2015

Protein purification can be one of the most stressful lab activities. Working with proteins requires a substantial amount of properly folded, relatively pure protein, but getting to this stage is often much easier said than done. As reviewed in our Plasmids 101 series, proteins are overexpressed from a plasmid construct, most often in special E. coli strains designed for protein expression. Cultures are then lysed and the protein of interest is purified using an affinity tag. Additional tags may be used to improve protein stability and solubility.

Determining the best way to express one’s protein of interest can save a lot of time later.

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Topics: Plasmid Tags, Plasmids

Plasmids 101: Protein tags

Posted by Eric J. Perkins on December 11, 2014

Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the accompanying cartoon, they have a multitude of uses including (but not limited to) purification, detection, solubilization, localization, or protease protection. Thus far Plasmids 101 has covered GFP and its related fluorescent proteins, which are sometimes used as tags for detection; however, those are just one (admittedly large) class of common fusion protein tags. Biochemists and molecular biologists who need to overexpress and purify proteins can face any number of technical challenges depending on their protein of interest. After several decades of trying to address these challenges, researchers have amassed a considerable molecular tool box of tags and fusion proteins to aid in the expression and purification of recombinant proteins.

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Topics: Plasmid Elements, Plasmids 101, Plasmid Tags

SpyLigase Irreversibly Locks Peptides Together for Efficient Cell Capture

Posted by Kendall Morgan on August 13, 2014

Mark Howarth’s lab at the University of Oxford is dedicated to generating new tools to manipulate biology based on molecular features found in nature, with the ultimate goal to improve the diagnosis of disease, and cancer in particular. They recently introduced the SpyTag/SpyCatcher system, based on a protein isolated from Streptococcus pyogenes that locks itself together, to produce irreversible protein-peptide interactions. In a study published in Proceedings of the National Academy of Sciences in March, he and his colleagues took another important step forward by dissecting that S. pyogenes protein into three parts. Their efforts yielded a protein, which they call SpyLigase (Spy comes from the “S” in Streptococcus and the “py” in pyogenes), capable of locking two peptide tags together.

SpyLigase overcomes limitations in the use of peptide tags, which often form only weak and reversible bonds. Howarth’s team has already demonstrated in their PNAS paper that SpyLigase can be used to link affibodies or antibodies against common tumor markers to subsequently capture cancerous cells expressing low levels of tumor antigen. I asked Howarth to tell us more about SpyLigase, its development, and its potential uses.

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Topics: Plasmid Tags, Plasmids

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