1. Epidermal Growth Factor Receptor (EGFR) the transmembrane + juxtamembrane domains
CR1 L2
CR2 JM
Kinase CT
644
151
312
481
621
687
955
1186
Extracellular portion
Intracellular portion
The transmembrane + juxtamembrane part ( a.a.) contains the transmembrane and the regulatory juxtamembrane (JM) domain. The construct carries N-terminal 7His-tag and C-terminal StrepII-tag (WSHPQFEK)

2. Important 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,

3. tj-EGFR: why two tags? Relation to the previous studies
Expression and purification
MALDI-TOF m
m His-blot SDS-PAGE 20 15 10 20 15
MHHHHHHHGPKIPSIATGMVGALLLLLVVAL GIGLFMRRRH IVRKR TLRR LLQERELVEPLTPSGEAPNQALLRILKETE
The results with tj-EGFR carrying ONLY 7His-tag were unsatisfactory

4. Expression of tj-EGFR in pET 27b+ E.coli BL21(DE3) Codon Plus RP
m b m b
m b m b 20 15 10 20 10
Strep-blot
His-blot
4 – 4 hours after induction 16 – 16 hours after induction 24 – 24 hours after induction
m – marker b – before induction red – at +37°C / blue – at + 28°C
The optimal expression conditions for tj-EGFR are:
+28°C, 24 hours

5. Purification of tj-EGFR in OG on Chelating and Strep-Tactin Sepharose
SDS-PAGE His-blot Strep-Blot
open cells
chelating Cu2+
Strep-Tactin
RPC 20 15 10
tj-EGFR can be purified to homogeneity

6. MALDI-TOF analysis of tj-EGFR
Only full-length tj-EGFR is observed:
no degradation products
(many thanks to Axel Niebisch, IBT-1)

7. CD spectra of tj-EGFR and secondary structure predictions: water and TFE
α-helix
β-sheet
turn
random
water
18%
29.5%
22.5%
30%
TFE
40%
13%
18.5%
28.5%

8. CD spectra of tj-EGFR and secondary structure predictions: detergents
50 mM NaP pH 6.0, 100 mM detergent
α-helix
β-sheet
turn
random OG
21%
28%
23%
SDS
29%
20%
30%
DPC
32%
27%
DHPC
25%
22%
LPPG
26%
19%

9. Sequence-based secondary structure predictions
MHHHHHHHGPKIPSIATGMVGALLLLLVVALGIGLFMRRRHIVR
KRTLRRLLQERELVEPLTPSGEAPNQALLRILKETEWSHPQFEK
~ 60% α-helix and ~ 40% random coil
There is a discrepancy between the experimental and predicted secondary structure structure content

10. What is yet to be done with tj-EGFR
15N / 13C structural NMR structural investigations
more detailed characterization by CD spectroscopy

11. THANKS FOR YOUR ATTENTION