Design of highly stabilized cold shock protein having high binding ability and its application for PCR method

2019 Fiscal Year Final Research Report

• Project/Area Number: 17K05926
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Bio-related chemistry
• Research Institution: Nagaoka University of Technology
• Principal Investigator: Kidokoro Shun-ichi 長岡技術科学大学, 工学研究科, 教授 (80195320)
• Project Period (FY): 2017-04-01 – 2020-03-31
• Keywords: 蛋白質核酸相互作用 / 分子設計 / 高安定化 / 一本鎖核酸 / PCR / 熱測定 / アニーリング / 熱解離

Outline of Final Research Achievements

Based on the three-dimensional structure of a cold shock protein (CSP) derived from Thermus thermophilus HB8, we designed a mutant CSP having two disulfide bonds, and expressed and purified it using Escherichia coli. Its thermal denaturation temperature above 100 C was observed at neutral pH and confirmed to have enough thermostability for PCR method, and to retain almost the same binding property as the wild type. Coexistence of the highly stabilized mutant at its appropriate concentration during PCR was shown to be effective in shortening the extension reaction time, but the presence of higher concentration of the mutant decreased the PCR product. DSC analysis indicated that it was mainly caused by the destabilizing effect of the CSP on the double stranded DNA structure formation and that it is important to design the mutant to show sharper thermal dissociation of the CSP-DNA complex for PCR application.

Free Research Field

蛋白質物性

Academic Significance and Societal Importance of the Research Achievements

本研究では、立体構造に基づく合理的設計により、分子機能を保持したまま１００℃を超える高い熱安定性を付与することに成功し、また蛋白質核酸相互作用の新しい熱力学的手法を提供しており、学術的な意義は大きい。また、PCR法は遺伝子を扱う最も基本的な技術の一つであり、最近のCOVID-19の感染判定に使用されるなど広く使用されているが、途中で生じる一本鎖核酸は、分子内・分子間で高次構造を形成しやすい性質を持ち、これがPCRの予期しない結果を生む主な原因と考えられる。本研究課題は、一本鎖核酸の高次構造形成を抑制することでこの問題の解決をめざし、この実現のための課題を明確にしており、社会的意義も大きい。