Aims Aims
In an attempt to crystallize all components of the type IV secretion system,
we have initiated a targeted structural genomics program which aims at cloning
and expressing genes encoding protein homologues of the type IV secretion system
across species. So far 7 organisms have been selected: Escherichia coli (pRP4,
pKM101), Agrobacterium tumefaciens, Helicobacter pylori, Bordetella pertussis,
Rickettsia prowazeki, Legionella pneumophila and Brucella suis. The table
below lists the genes encoding the components of type IV secretion systems in
these organisms.
|
strains
|
identified genes
|
pathologies
|
| Escherichia coli (pRP4, pKM101) | traA,B,C,D,E,F,G,L,M,N,O,korA,eex | |
| Agrobacterium tumefaciens | virB1,B2,B3,B4,B5,B6,B7,B8,B9,B10,B11,virD4 | plant tumor |
| Helicobacter pylori, | cag7,8,9,12,23,virB11 | gastric ulcer |
| Bordetella pertussis | ptlA,B,C,D,E,F,G,H,I | whopping cough |
| Rickettsia prowazeki | ORF103,287,290,291,292,293 | typhus |
| Legionella pneumophila | icmE,dolE | pneumonia |
| Brucella suis | virB1,B2,B3,B4,B5,B6,B7,B8,B9,B10,B11 | brucellosis |
Strategy
|
|
A SummaryB Detailed
description C Biocomputing
approachesD Experimental
approaches |
Our strategy is as follows:
|
|
|
B1-
Target selection & protein-protein interaction mapping
Selection
of protein sequence targets by database mining of selected genomes
Selection
of proteins encoded by pathogenicity islands or other gene clusters
Detection
of potential partners by Rosetta stone mining
Detection
of potential partners by phylogenetic profiles
B2-
Prediction of protein fragment solubility
Detection
of signal peptides (SignalP program)
Detection
of transmembrane regions (TMHMM program)
B3-
Selection of oligonucleotides for cloning
Oligonucleotide
design used to clone genes encoding soluble protein fragments
B4-
Cloning by PCR in TOPO TA vectors
Amplification
of gene fragments from bacterial
DNA by PCR
Cloning
of amplified
DNA fragments into a TOPO TA vector
Test
of insertion and gene orientation by PCR
B5-
Expression in E.coli
Transformation
of E.coli strain BL21 DE3 with TOPO TA vector
Cultures
in LB (amp, chm), induction by IPTG
B6-
Purification on Ni column (1st step)
Bacteria
sonicated and centrifugated, separation of cytoplasmic, membrane and inclusion
body fractions.
Solubilization
of the selected fraction and loading on Ni-column,
washing and elution
B7-
Purification by gel exclusion chromatography or ion exchange chromatography
(2sd step)
Purification
from eluted fractions by gel-filtration or ion-exchange column
B8-
Control using SDS-PAGE, mass-spectrometry and light scattering
SDS-PAGE
Molecular
weight by mass spectrometry
Molecular
species identification by light-scattering / gel filtration
B9-
Concentration and buffer exchange
Concentration
through spin filters and buffer exchange
Buffer
exchange by dialysis if needed
B10-
Crystallization trials of individual molecules
Exploration
of crystallization conditions on 24-wells plates using the hanging drop method
B13-
Protein association scanning
B14-
Structure analysis of complexes
protein
cocrystallisation
electronic
microscopy
B15-
Cloning, expression, purification, and crystallization of membrane proteins
B16-
Structure analysis of membrane-associated complexes
B17-
Structure validation and depositions
C-Biocomputing
approaches
Biocomputing
protocoles
Biocomputing
results
D- Experimental
approaches (in progress)
Experimental
protocoles
Experimental
results
Experimental
database
| Coordinator Gabriel Waksman . |
Last modification : 12/22/2000 by Thierry
Rose .
|