Development of single molecule display platform for directed evolution of enzymes

2018 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: mRNA/cDNA display / directed evolution / protein engineering / in vitro transcription / in vitro translation / peroxidases

Outline of Annual Research Achievements

In the previous year (FY 2018) I was on a maternity leave for 6 months (June-December), and my original plan for research got delayed. I was able to do a part of the preparations.
We optimized preparation of RNA library; subcloned horseradishperoxidase (HRP) (w/o solubilization tags) to investigate the improvement of HRP's solubility during expression in an E.coli-based expression system. HRP constructs were co-expressed with Erv1/cDsbc chaperones. HRP with T7 tag had the highest level of soluble peroxidase. Its activity can be reconstituted by incubation with its cofactor.
This finding is important for the directed evolution of peroxidases by the new platform, since we need to make sure that the peroxidase can be expressed as active protein by the E.coli cell-free protein synthesis system.

Current Status of Research Progress

4: Progress in research has been delayed.

Reason

The main reason is my absence from work due to maternity (child care) leave (for 6 months, from June until December 2018).

Strategy for Future Research Activity

The research will be continued as planed (in the order outlined in the proposal).
I may engineer more protein/peptide targets by the new platform, if the time is sufficient.

Causes of Carryover

In the FY2019 I need to do main part of the research plan (establish the platform for directed evolution of peroxidases; originally planed for FY2018 but delayed due to my maternity leave), thus I need funds to purchaes consumables and present my research data at national and international conferences.