Epidermal%20Growth%20Factor%20Receptor%20(EGFR)%20the%20transmembrane%20 %20juxtamembrane%20domains

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Epidermal 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)
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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, 24043-52

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tj-EGFR why two tags?Relation to the previous
studies
Expression and purification
MALDI-TOF
m 1 2 3 4
m His-blot SDS-PAGE
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15
MHHHHHHHGPKIPSIATGMVGALLLLLVVAL GIGLFMRRRH IVRKR
TLRR LLQERELVEPLTPSGEAPNQALLRILKETE
The results with tj-EGFR carrying ONLY 7His-tag
were unsatisfactory
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Expression 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
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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
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Purification of tj-EGFR in OG on Chelating and
Strep-Tactin Sepharose
SDS-PAGE His-blot Strep-Blot
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15
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tj-EGFR can be purified to homogeneity
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MALDI-TOF analysis of tj-EGFR
Only full-length tj-EGFR is observed no
degradation products
(many thanks to Axel Niebisch, IBT-1)
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CD 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
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CD 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
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Sequence-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
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What is yet to be done with tj-EGFR

• 15N / 13C structural NMR structural
  investigations
• more detailed characterization by CD spectroscopy

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THANKS FOR YOUR ATTENTION