Realization of eukaryotic synthesis speed in Escherichia coli by cassette exchange of ribosome stalk complex

2015 Fiscal Year Final Research Report

• Project/Area Number: 26650013
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Structural biochemistry
• Research Institution: Hokkaido University
• Principal Investigator: YAO Min 北海道大学, 先端生命科学研究科(研究院), 教授 (40311518)
• Co-Investigator(Renkei-kenkyūsha): UCHIUMI Toshio 新潟大学, 大学院自然科学研究科, 教授 (50143764) ; KATO Koji 北海道大学, 大学院先端生命科学研究院, 助教 (30452428)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Keywords: 翻訳速度 / 発現系 / ストーク複合体 / タンパク質工学 / リボソーム / X線結晶解析

Outline of Final Research Achievements

The protein synthesis speed of ribosome is one of the important factors for over-expressing soluble protein using Escherichia coli, especially over-expressing eukaryotic proteins. In addition, it has been known that the bacterial protein synthesis speed (GTPases-turnover) is about 10 times of eukaryote. In order to construct an over-expression system with low speed of protein synthesis (translation) of E. coli, we tried to modify ribosomal stalk L10 from E. coli to a chimera L10 (L10P0) which binds to both ribosomal protein L12 and P1 (eukaryotic type), and has a chimera characters of bacteria and eukaryote of GTPases-turnover. We have successfully expressed mutants of L10ΔCH, L10ΔCH-P0H2CTD, and L10ΔCH-P0H3CTD. The binding assay of purified L10ΔCH-P0H2CTD with P1 showed the possibility for constructing a chimera stalk complex which will reduce the protein synthesis speed on ribosome.

Free Research Field

構造生物学，タンパク質結晶学