Development of a system for enhancing protein thermostability in vivo using hyperthermophiles
| Project/Area Number |
15350100
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Chemistry related to living body
|
|---|
| Research Institution | Kyoto University |
|---|
Principal Investigator |
ATOMI Haruyuki Kyoto University, Graduate School of Engineering, Associate Professor, 工学研究科, 助教授 (90243047)
|
|---|
| Project Period (FY) |
2003 – 2004
|
| Project Status |
Completed (Fiscal Year 2004)
|
| Budget Amount *help |
¥15,200,000 (Direct Cost: ¥15,200,000)
Fiscal Year 2004: ¥5,800,000 (Direct Cost: ¥5,800,000)
Fiscal Year 2003: ¥9,400,000 (Direct Cost: ¥9,400,000)
|
|---|
| Keywords | Hyperthermophilic archaea / gene integration / gene complement / unfolded protein response / chaperonin / pyrF / uracil / Thermococcus / in vivo進化 / 超好熱菌 / 始原菌 / タンパク質の耐熱化 / 熱安定性 / 分子進化 / chaperonin HSP60 / Directed evolution |
|---|
| Research Abstract |
We have been examining whether homologous genes from other archaeal strains could complement endogenous gene disruption in T.kodakaraensis and whether proteins with less thermostability could be subject to directed evolution in terms of increasing thermostability. T.kodakaraensis KUW1 is a strain whose pyrF and trpE genes are deleted. To this strain, we introduced pyrF genes from the hyperthermophilic archaeon Pyrococcus furiosus (optimum growth temperature 100℃) or the thermophilic archaeon Thermoplasma volcanium (optimum 60℃) via homologous recombination. We found that the recombinant strain with the P. furiosus pyrF gene exhibited uracil prototrophy, indicating that gene complementation had occurred. Similar results were obtained with the gene from T volcanium, however, consistent growth was observed only at temperatures at or below 75℃. Intriguingly, at 75℃, we found an intracellular accumulation of CpkB in this strain, but not in the wild type T.kodakaraensis KOD1 or in the strain harboring P.furiosus pyrF. As the 75℃ temperature is well below the optimal growth temperature of T.kodakaraensis, the accumulation of CpkB cannot be due to thermal stress. This raise the possibilities that CpkB expression is responding to the intracellular presence of partially denatured PyrF protein from T volcanium, a mechanism which corresponds to the unfolded protein response observed in bacteria and eukaryotes.
|
Report
(3 results)
Research Products
(20 results)
