mem-iLID, a fast and economic protein purification method

Abstract

Protein purification is the vital basis to study the function, structure and interaction of proteins. Widely used methods are affinity chromatography-based purifications, which require different chromatography columns and harsh conditions, such as acidic pH and/or adding imidazole or high salt concentration, to elute and collect the purified proteins. Here we established an easy and fast purification method for soluble proteins under mild conditions, based on the light-induced protein dimerization system iLID, which regulates protein binding and release with light. We utilize the biological membrane, which can be easily separated by centrifugation, as the port to anchor the target proteins. In Xenopus laevis oocyte and Escherichia coli, the blue light-sensitive part of iLID, AsLOV2-SsrA, was targeted to the plasma membrane by different membrane anchors. The other part of iLID, SspB, was fused with the protein of interest (POI) and expressed in the cytosol. The SspB-POI can be captured to the membrane fraction through light-induced binding to AsLOV2-SsrA and then released purely to fresh buffer in the dark after simple centrifugation and washing. This method, named mem-iLID, is very flexible in scale and economic. We demonstrate the quickly obtained yield of two pure and fully functional enzymes: a DNA polymerase and a light-activated adenylyl cyclase. Furthermore, we also designed a new SspB mutant for better dissociation and less interference with the protein of interest, which could potentially facilitate other optogenetic manipulations of protein-protein interaction.

Optogenetic Methods in Plant Biology

“…Several additional optogenetic applications likely to be introduced into plants include the manipulation of cellular processes by allosteric regulation of protein activity and/or interactions, using LOV-Jα-based tools (154) or light-induced dimer pairs, for example, PHYB/PIF (84,85,135,144), CRY2/CIB1 (73), iLID (54), and TULIPs, in addition to dozens of photoswitches (https://www.optobase.org/). Such tools can be implemented for, among other things, the spatiotemporal regulation of protein activity (92, 154), the subcellular targeting of endogenous proteins (44,157), the induction of phase separation processes (14), protein purification (63,143), and the subcellular enhancement of protein activities (9,10). Furthermore, tissue-specific and subcellular application of different tools will help to probe plant responses at a defined cellular or even subcellular level.…”

1Progress, applications, challenges and prospects of protein purification technology

“…In recent years, with the completion of genome sequencing of many species ( Shendure and Lieberman Aiden, 2012 ; Beigh, 2016 ; Park and Kim, 2016 ; Ing-Simmons and Vaquerizas, 2019 ), researches in protein related fields have gradually become the focus of the biotechnology industry ( Labrou, 2021 ), and many purification technologies have been gradually applied from protein and enzyme separation to drug synthesis, vaccine research and development, clinical detection, environmental analysis and biophysical measurement ( Pfaunmiller et al, 2013 ). Therefore, high-quality protein samples are of great significance for the subsequent structure and function research and related products development ( Tang et al, 2021 ). However, proteins have the characteristics of complex structure, easy to be affected by internal and external factors, easy to be degraded, etc.…”