Production of recombinant full-length membrane-spanning vacuolar sorting receptor of soybean and complex formation with cargo protein

Research Project

Project/Area Number	20K22564
Research Category	Grant-in-Aid for Research Activity Start-up
Allocation Type	Multi-year Fund
Review Section	0601:Agricultural chemistry and related fields
Research Institution	Kyoto University
Principal Investigator	Cabanos Cerrone Salamat 京都大学, 農学研究科, 准教授 (50875474)
Project Period (FY)	2020-09-11 – 2022-03-31
Project Status	Completed (Fiscal Year 2021)
Budget Amount *help	¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000) Fiscal Year 2021: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000) Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Keywords	sorting receptor / vacuole / seed storage protein / soybean / Seed storage protein / Vacuole / Sorting Receptor / Vacuolar Receptor
Outline of Research at the Start	I. Develop a yeast expression system for the full-length soybean vacuolar sorting receptor (VSR). II. Develop a protocol for VSR purification and optimize protein yield. III. Optimize protocol for complex formation between the purified full-length GmVSR and its known seed storage cargo protein
Outline of Final Research Achievements	Using codon optimized constructs, I transformed and optimized expression of the full-length soybean vacuolar sorting receptor in yeasts, S. cerevisiae and Pichia pastoris. Although GmVSR in Pichia had higher expression level than Saccharomyces, the yield was very low for use in structural elucidation. I then designed a minimal construct with luminal region only (GmVSRlum) containing essential binding domains. GmVSRlum showed promising yield, however, upon testing for cargo binding, it did not show any binding at all. Instead of 3D determination by X-ray crystallography, I carried out homology modeling via RaptorX and AlphaFold2 to obtain its predicted 3D model and, based on this, propose that residues R102, F106, D136, E139 contribute to soybean VSR-cargo interaction. Here, I was not able to establish the yeast expression system suitable for soybean VSR. In the future, I surmise that mutagenesis in the protein combined with molecular dynamics simulations might help solve this problem.
Academic Significance and Societal Importance of the Research Achievements	The results of this study will serve as a stepping-stone towards elucidating the structure and binding mechanism of VSR to its cargo. The findings of the study will also help expand and transform protein sorting biology and could bring insight into the development of protein-rich crops.