2021 Fiscal Year Annual Research Report
Production of recombinant full-length membrane-spanning vacuolar sorting receptor of soybean and complex formation with cargo protein
| Project/Area Number |
20K22564
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| Research Institution | Kyoto University |
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Principal Investigator |
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| Project Period (FY) |
2020-09-11 – 2022-03-31
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| Keywords | Seed storage protein / Vacuole / Sorting Receptor / Vacuolar Receptor |
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| Outline of Annual Research Achievements |
Vacuolar sorting receptors are responsible for recognizing cargo-proteins and transporting them to vacuole - a crucial process in plant survival. However, underlying mechanism of VSR-cargo binding remains elusive. Due to technical difficulty in expressing and purifying membrane proteins, no study has ever worked on full-length VSR. Here, I attempted to develop a cost-effective yeast expression system and purification protocol for full-length membrane-spanning vacuolar sorting receptor. Using codon optimized constructs of soybean VSR, I transformed and optimized expression in yeasts, S. cerevisiae and Pichia pastoris. Although VSR in Pichia had the highest expression level, the yield was still very low for use in structural elucidation. I then designed a minimal construct with only the luminal region (VSRlum) containing the essential binding domains. Expression of VSRlum has been previously done in albeit costly baculovirus-insect cell system. Compared to full-length VSR, VSRlum showed promising yield. However, upon testing its binding to cargo, the luminal construct did not show any binding at all. Instead of 3D determination by X-ray crystallography, I carried out VSRlum structure homology modeling via RaptorX and AlphaFold2 to obtain the predicted 3D model and, based on this, propose that residues R102, D136, E152 and F106 contribute to soybean VSR-cargo or ligand interaction. In this study, I was not able to establish the yeast expression system suitable for soybean VSR. Random mutagenesis in combination with molecular dynamics simulations might help solve this problem.
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