Development of single molecule display platform for directed evolution of enzymes

2019 Fiscal Year Research-status Report

• Project/Area Number: 18K14387
• Research Institution: Nagoya University
• 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)
• Project Period (FY): 2018-04-01 – 2022-03-31
• Keywords: cDNA display / directed evolution / protein engineering / in vitro translation

Outline of Annual Research Achievements

In AY2019, I changed the target of my experiment from peroxidase to a shorter peptide, T26, the substrate of an enzyme called transglutaminase. It seemed difficult to work with peroxidase due to its poor expression yield, so we decided to start with easier target to establish the platform. I used T26 peptide fused with GST tag, as previously reported by another group to have good expression yield in E.coli. As a negative control, I used mutated T26 peptide which is not a substrate of transglutaminase. We screened 1:1, 1:10, 1:100 and 1:1000 (= T26:T26N) binary libraries. The enrichment ratio of T26 peptide looked very good for 1:1 library, however, as the amount of T26N increased in the mixture, the enrichment ratio worsened. To solve this problem, I tried changing various conditions during the selection step and afterwards. Since I could not get the desired result, we decided to try changing the fusion partner of T26. The current fusion partner is GST, however, this protein forms dimers and has free Cysteine residues, both of which could cause interference with the selection step, or loss of the displayed peptides due to aggregation. Thus we decided to replace it with luciferase (NanoLuc), or GFP (green fluorescent protein). We are also investigating whether T26 could be displayed without any protein tag. The new gene constructs have been made, and are under experimental testing now. We have also changed the linker used in the study to connect the nucleic acids and protein to the one which requires less time for the crosslinking, to avoid exposure of mRNA to degradation.

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

I had to change the target of my experiment, i.e. choose a different protein, since the initial choice did not work well for the purpose of my experiment. This is where I lost considerable time. Another problem was the optimization of the selection step to get the satisfactory enrichment. This step also took a lot of time, before I realized change of the fusion partner could be necessary.

Strategy for Future Research Activity

In AY2020, I plan to finalize the screening of binary libraries of T26: T26N peptide, and conduct semi-random T26 library screening followed by next-generation sequencing. If the screening of binary libraries is successful, we can consider that the platform is established and proceed with the semi-random library screening to discover the new T26 peptide sequences preferred by the transglutaminase. As for the second part of the project where I planed to use peroxidase as a reporter module for engineering of other enzymes, I recently found a report about another plant peroxidase, soybean ascorbate peroxidase (APEX2), which can be successfully expressed in E.coli. The expression vector is commercially available, and I plan to obtain it and use APEX2 as a reporter enzyme for engineering of other enzymes, as described in my original proposal. If necessary, APEX2 can also be engineered to improve its properties, as described before.

Causes of Carryover

Due to the global health crisis, I cannot travel and attend international conferences this year. In fact, I am not sure if I can attend local meetings either. However, I included participation in at least one local scientific meeting (if circumstances allow). This is why I changed "travel expenses" part. Since my progress is slightly delayed and I work on the project by myself, I decided it would be better to hire an assistant to help me speed up the work and finish the planed experiments sooner. That is the reason why I included "personal expenditure and remuneration expenses". The assistant will be hired for three months because I am not sure if I can afford to hire her for longer period of time. The rest of the budget will be used to obtain chemicals necessary for experimental work. The burden on this part of the budget ("article costs") has been alleviated a bit because we started collaborating with GeneFrontier Corporation, the provider of in vitro translation kits, so that we can receive these kits for free.