2021 Fiscal Year Final 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 Category |
Grant-in-Aid for Research Activity Start-up
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| Allocation Type | Multi-year Fund |
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| Review Section |
0601:Agricultural chemistry and related fields
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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 | sorting receptor / vacuole / seed storage protein / soybean |
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| 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.
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| Free Research Field |
applied agricultural biochemistry
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| 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.
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