2020 Fiscal Year Research-status 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 |
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| Outline of Annual Research Achievements |
I prepared codon optimized constructs of soybean full-length VSR. I transformed and optimized its expression in budding yeast, S. cerevisiae and methylotrophic yeast, Pichia pastoris as host. VSR in Pichia had the highest expression level. However, despite optimization, the protein expression and yield is very low. I then designed truncated constructs containing the luminal domain that is essential for binding to protein cargo. Expression showed at least five-fold higher than the full-length construct. It can be purified to homogeneity with a yield that is suitable for further structural characterization and binding with protein cargo.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Truncating the protein proved helpful in increasing protein yield. Although truncated, the final protein still contains the essential domains necessary for cargo binding and useful in answering the ultimate goal of the project.
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| Strategy for Future Research Activity |
I plan to proceed with the purification of the intact protein cargo (soybean 7S globulin) and optimize the conditions for VSR-protein cargo complex formation that is suitable for cryo-EM elucidation. I will also further optimize large-scale purification conditions to obtain much higher yield for the VSR protein. Interaction and binding kinetics will also be determined.
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