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X-ray crystallography of the transcriptional activator PhoB from Escherichia coli

Research Project

Project/Area Number 09680662
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Biophysics
Research InstitutionNational Institute of Genetics

Principal Investigator

AKIBA Toshihiko  National Institute of Genetics, Structural Biology Center, research associate, 構造遺伝学研究センター, 助手 (80291889)

Project Period (FY) 1997 – 1998
Project Status Completed (Fiscal Year 1998)
Budget Amount *help
¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1998: ¥600,000 (Direct Cost: ¥600,000)
KeywordsPhoB protein / transcriptional activator / DNA-binding protein / crystallization / X-ray crystallography / protein over-expression / column chromatography / protein purification / リン酸化 / 転写制御 / 二成分制御系
Research Abstract

1. The C-terminal DNA-binding domain of PhoB has been overexpressed and purified to homogeneity as judged by overloaded silver-stained SDS-PAGE gels by an improved method including (1) precipitation of nucleic acid by protamine sulfate, (2) heparin affinity chromatography, and (3) cation exchange chromatography operated in a pH gradient mode.The purified protein fragment was crystallized in the same condition as reported previously.Trigonal crystals appeared after one month of incubation.This growth speed is three-times faster than that of crystals grown from protein solutions prepared by the older methods.The crystals were soaked in K_2PtCl_4 solution to produce heavy atom derivatives, from one of which X-ray diffraction patterns were collected.
2. Induction of overexpression of intact PhoB protein was frequently failed, presumably due to loss of protein-expressing cells.To circumvent this problem, we have devised two procedures ; (1) protein production by leaky cells in the absence of an inducer, and (2) use of solid medium for preculture and use of more stringent antibiotics.Yield of the protein was 10 mg/L in the best case.
3. Intact PhoB protein has been purified to homogeneity as judged by overloaded CBB-stained SDS-PAGE gels by the newly developed method, which includes (1) precipitation of nucleic acid by protamine sulfate, (2) anion exchange chromatography, (3) heparin affinity chromatography, and (4) cation exchange chromatography operated in a pH gradient mode.
4. Crystallization conditions of intact PhoB protein have been searched by a micro-dialysis method and a micro-sitting drop method.The latter method was more useful in the present case where supply of the protein was limited.The best result obtained so far is a microcrystal-like granular precipitation produced by ammonium sulfate.Spherulites were obtained in a few examples of crystallization in the presence of acetylphosphate and Mg^<++> which induce in vitro phosphorylation of the protein.

Report

(3 results)
  • 1998 Annual Research Report   Final Research Report Summary
  • 1997 Annual Research Report

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Published: 1998-04-01   Modified: 2016-04-21  

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