2025/11/01 by Scott Schwartz, Carina Engstler, Susanne Mühlbauer +4 · 1 voice · 2 citations
Biochemistry, Genetics and Molecular Biology · Chemistry · #Transgenic Plants and Applications #Biochemical and Structural Characterization #Advanced Proteomics Techniques and Applications
paper · pdf · doi:10.1111/tpj.70544
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, multistep 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 proteins from plant tissues. The Catch & Release toolbox includes a vector set, a homemade GFP-Trap, and homemade proteases. We demonstrate how the Catch & Release vectors streamline cloning and transgenic plant selection through the fluorescence-accumulating seed technology, 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 by complementation and transient protein expression experiments. We further show that besides facilitating standard molecular biological experimentation, fusion proteins expressed using our vector set in combination with homemade GFP-Trap and proteases enable efficient and rapid isolation of enzymatically active, soluble, and high molecular weight membrane proteins directly from plants. By following our detailed reagent preparation instructions, purification costs can be decreased up to 60-fold compared with commercially available options.