Catch & Release – rapid cost-effective protein purification from plants using a DIY GFP-Trap-protease approach
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Abstract
The purification of proteins is the foundation to study their structure, function, biochemical properties, and interaction partners. In plant research, unique challenges arise from the complexity of plant tissues, interference of secondary metabolites, and sometimes the low abundance of target proteins. Many conventional plant protein purification methods rely on expensive reagents, multi-step procedures, and labor-intensive workflows, limiting their feasibility for many applications. Here, we present the “Catch & Release” system, a cost-effective, fast and reliable one-step purification workflow for the isolation of soluble and membrane-bound proteins from plant tissues. The Catch & Release toolbox includes a vector set, a homemade GFP-trap and homemade proteases. Catch & Release vectors streamline cloning and transgenic plant selection through the Fluorescence-Accumulating Seed Technology (FAST), which marks positive transformants with a strongly fluorescing seed coat. Each plasmid consists of four, easy to exchange, modules: a plant promoter, a cloning dropout marker, protease cleavage sites, and seven different epitope tags, including an innovative dual-fluorescent tag, providing flexibility for diverse experimental needs. The in vivo functionality of all modules has been confirmed. Besides enabling standard molecular biological experimentation, our vector set in combination with homemade GFP-trap and proteases enables efficient and rapid isolation of soluble and high molecular weight membrane proteins directly from plants. By following our detailed reagent preparation instructions, purification costs can be decreased hundred-fold compared to the commercially available options.
Article activity feed
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activity measurements
It's really awesome that you developed these tools and processes to enable protein production in plants!
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enzymatically functional
I wonder if you could also integrate a DSF or nano-DSF protocol to confirm folding/thermal stability of your protein as well. It could be another good indicator of successful protein production.
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∼100 µg protein/g
That's great!
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supernatant
this is a cool feature!
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starting material
Given this is a membrane protein, did you have issues with solubility? Do you have data showing the protein is well folded after purification?
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GFP-trap
How long is this stable for? Do you have to make it fresh for each purification you do?
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system
Are you treating with TEV in whole plant lysate?
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co-localization of mCherry and mVenus
I am not sure that co-localization of those tags is the best way to confirm that the protein integrity contains both tags. It is unlikely that you could see single proteins based on your image resolution - I think the stronger claim is the western blot you have that shows the size change upon cleavage (fig 2E,D)- and also just the fact that your protein is of expected size with the two tags.
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fusion tag
It is super helpful that you included so many tag designs already in the construct. It really adds to the usability right off the bat.
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