Screening of a minimal reporter protein with correct SS-bonds using a novel VanX mediated E-coli autolysis protocol

2015 Fiscal Year Final Research Report

• Project/Area Number: 26560432
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Biomolecular chemistry
• Research Institution: Tokyo University of Agriculture and Technology
• Principal Investigator: Kuroda Yutaka 東京農工大学, 工学(系)研究科(研究院), 准教授 (10312240)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: 蛋白質工学 / 生物活性物質の探索 / SS結合 / 探索法 / ランダム変異

Outline of Final Research Achievements

Gaussia Luciferase (GLuc; 168 aa) is among the smallest luciferase and could serve as a reported protein in biochemical research. Its identification is relatively recent, and its structure and biophysical properties remain to be fully characterized. Here, we developed a novel method for screening GLuc, where VanX is coexpressed with Gluc in order to autolyse E-coli and release GLuc into the culture media. This, in turn, enables GLuc’s bioluminescence measurement directly in the crude media without purification greatly increasing the screening efficiency. Using this protocol we identified several single mutation GLuc variants with light emission red shift by up to 5 nm, and a double mutation variant with red shift of 15 nm (but with weaker light emission intensity). In addition to this, we started a heteronuclear NMR analysis of recombinant GLuc, and we assigned about 70% of its main chain peak and their secondary structures.

Free Research Field

生物物理・蛋白質の物理化学