2020 Fiscal Year Research-status Report
Development of single molecule display platform for directed evolution of enzymes
| Project/Area Number |
18K14387
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| Research Institution | Nagoya University |
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Principal Investigator |
ダムナニョヴィッチ ヤスミナ 名古屋大学, 生命農学研究科, 助教 (00754673)
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| Project Period (FY) |
2018-04-01 – 2022-03-31
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| Keywords | cDNA display / transglutaminase / library screening / NGS / bioinformatics |
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| Outline of Annual Research Achievements |
In AY2020, I aimed at developing a platform coupling the advantages of cDNA display with high-throughput and accuracy of next-generation sequencing for the selection of preferred transglutaminase 2 substrate peptides. After the optimization of the platform by the repeated screening of binary model libraries, one round of screening and selection of fully randomized four-amino-acid library at positions -1, +1, +2, and +3 from the glutamine residue was carried out. Enriched cDNA complexes were analyzed by next-generation sequencing and bioinformatics, revealing the comprehensive amino acid preference of the transglutaminase 2 in each position of the peptide backbone. With the help of bioinformatics, I obtained data on enrichment ratios of 20 amino acids at each randomized position after only one round of screening.
This platform represents a time-, labor- and cost-efficient tool for comprehensive analysis of TG substrate preference and holds promise for wide use in the development of TG peptide probes. The manuscript on this work is under submision to a scientific journal for publication.
I have also started establishment of the cDNA display platform with engineered peroxidase as a reporter enzyme, for engineering of D-amino acid oxidase (DAAO) and phospholipase D, as originally planed. We are now investing cell-free protein synthesis conditions of DAAO, and its display on the puromycin linker.
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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
The progress for AY2020 is as expected given the difficulties we all faced due to the corona virus infection risk. In spite of having to frequently stop and resume my work, the research results came as desired.
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| Strategy for Future Research Activity |
1) Use the established platform for screening and selection of preferred transglutaminase substrate peptides to screen substrate preference of other transglutaminases (transglutaminase 1, important for medical research and microbial transglutaminase, important for biotechnological research). This work has already started in collaboration with Prof. Hitomi Kiyotaka (Nagoya University).
2)Use engineered soybean peroxidese, APEX2, as a reporter enzyme to screen mutant enzyme library and isolate variants with improved properties. This work is ongoing and I am focusing on mutating D-amino acid oxidase from yeast to change its substrate specificity. (According to the original research proposal)
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| Causes of Carryover |
I will need funds for AY2021 to finish the projects I am working on. The funds will be used to purchase reagents for the experiments, pay participation at national and international conferences (1 international (online) and at least 1 national conference are planed for AY2021). Possibly, I would like to hire an assistant for a few hours per week to help me with experiment preparations and increase efficincy of my work.
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