Title: Epidermal%20Growth%20Factor%20Receptor%20(EGFR)%20the%20transmembrane%20 %20juxtamembrane%20domains
1Epidermal Growth Factor Receptor (EGFR)the
transmembrane juxtamembrane domains
The transmembrane juxtamembrane part (615-686
a.a.) contains the transmembrane and the
regulatory juxtamembrane (JM) domain. The
construct carries N-terminal 7His-tag and
C-terminal StrepII-tag (WSHPQFEK)
2Important information about the tj-EGFR
- 73 amino acid residues (without tags)
- carries N-terminal 7His-tag (HHHHHHH)
- carries C-terminal StrepII-tag (WSHPQFEK)
- molecular weight is about 10,152 Da
- pI is around 11.2
- contains no Cys residues
- NMR structure of the juxtamembrane
- domain is available
- Choowongkomon et al. (2005), J. Biol. Chem.,
280, 24043-52
3tj-EGFR why two tags?Relation to the previous
studies
Expression and purification
MALDI-TOF
m 1 2 3 4
m His-blot SDS-PAGE
20
15
MHHHHHHHGPKIPSIATGMVGALLLLLVVAL GIGLFMRRRH IVRKR
TLRR LLQERELVEPLTPSGEAPNQALLRILKETE
The results with tj-EGFR carrying ONLY 7His-tag
were unsatisfactory
4Expression of tj-EGFR in pET 27b E.coli
BL21(DE3) Codon Plus RP
m b 4 16 24 m b 4 16 24
m b 4 16 24 m b 4 16 24
20
10
Strep-blot
His-blot
4 4 hours after induction16 16 hours after
induction24 24 hours after induction
m markerb before inductionred at 37C /
blue at 28C
The optimal expression conditions for tj-EGFR
are 28C, 24 hours
5Purification of tj-EGFR in OG on Chelating and
Strep-Tactin Sepharose
SDS-PAGE His-blot Strep-Blot
20
15
10
tj-EGFR can be purified to homogeneity
6MALDI-TOF analysis of tj-EGFR
Only full-length tj-EGFR is observed no
degradation products
(many thanks to Axel Niebisch, IBT-1)
7CD spectra of tj-EGFR and secondary structure
predictions water and TFE
a-helix ß-sheet turn random
water 18 29.5 22.5 30
TFE 40 13 18.5 28.5
8CD spectra of tj-EGFR and secondary structure
predictions detergents
50 mM NaP pH 6.0,100 mM detergent
a-helix ß-sheet turn random
OG 21 28 23 28
SDS 21 29 20 30
DPC 32 21 20 27
DHPC 23 25 22 30
LPPG 25 26 19 30
9Sequence-based secondary structure predictions
MHHHHHHHGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVR KRTLR
RLLQERELVEPLTPSGEAPNQALLRILKETEWSHPQFEK
- 60 a-helix and 40 random coil
There is a discrepancy between the experimental
and predicted secondary structure structure
content
10What is yet to be done with tj-EGFR
- 15N / 13C structural NMR structural
investigations - more detailed characterization by CD spectroscopy
11THANKS FOR YOUR ATTENTION