Abstract
Heterologous expression and purification of transmembrane proteins have remained a challenge for decades hampering detailed biochemical and structural characterization of key enzymes and their interacting regulators in multiple metabolic pathways. An in-depth study on the newly identified Arabidopsis thaliana integral membrane protein BALANCE OF CHLOROPHYLL METABOLISM 1 (BCM1) showed a stimulatory effect of the BCM1 on magnesium chelatase, the first enzyme of chlorophyll biosynthesis, through interaction with the GENOMES UNCOUPLED 4 (Wang et al., 2020). Here, we report a detailed and optimized method for heterologous expression and purification of His-tagged BCM1 in Saccharomyces cerevisiae. Following this method, we obtained native BCM1 used for in vitro enzymatic assay of magnesium chelatase (Wang et al., 2020). Currently, the crystallization studies of the BCM1 are underway. This protocol could be adapted to purify BCM1-like transmembrane proteins from eukaryotic organisms for enzymatic and structural studies.
Keywords: A transmembrane protein, Expression and purification of BCM1, Saccharomyces cerevisiae, Arabidopsis thaliana
Background
Identification of post-translational regulators which directly modulate the enzymatic activities of chlorophyll synthesis enzymes can greatly improve our understanding of molecular mechanisms, by which plants maintain highly efficient chlorophyll biosynthesis during leaf greening (Brzezowski et al., 2015). However, the detailed biochemical analyses of chlorophyll synthesis enzymes and their interacting proteins have been restricted by the availability of recombinant proteins in vitro. We recently identified a post-translational regulator BALANCE OF CHLOROPHYLL METABOLISM 1 (BCM1), which simultaneously stimulates chlorophyll biosynthesis and delays chlorophyll breakdown, thereby conferring chlorophyll homeostasis during leaf development (Wang et al., 2020). To examine BCM1’s effect on the enzymatic activity of magnesium chelatase (MgCh), the first enzyme of chlorophyll biosynthesis, we expressed and purified His-tagged BCM1 in S. cerevisiae. It has been shown that BCM1 is able to stimulate MgCh activity in vitro (Wang et al., 2020). Because BCM1 has six transmembrane domains, BCM1 will be used an example of multiple-pass protein herein. Thus, we provide an optimized method for expression and purification of BCM1 in S. cerevisiae. Although prokaryotic purification system hosted by Escherichia coli has been widely used to express hydrophilic proteins from prokaryotic and eukaryotic organisms, overexpression of BCM1 in Escherichia coli cells leads to accumulation of BCM1 aggregate and inclusion bodies instead of properly folded proteins at the membranes. In comparison with cell-free protein expression systems and other eukaryotic expression systems, such as mammalian and insect expression systems, the yeast protein expression method described here enables a large-scale purification of integral membrane proteins with high yield and low cost.
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Acknowledgments
This work was funded by the Alexander von Humboldt Foundation to P.W., and the Deutsche Forschungsgemeinschaft to P. W. (WA 4599/2-1), and to B.G. (FOR2092, GR 936/18-1 and SFB TR175, subproject C04). This protocol was adapted from a previously described method (Wang et al., 2020).
Competing interests
We declare no conflicting or competing interests.
References
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