1 Epidermal Growth Factor Receptor (EGFR) the transmembrane + juxtamembrane domains L1CR1L2CR2 JM KinaseCT Extracellular portionIntracellular portion L1CR1L2CR2 JM KinaseCT Extracellular portionIntracellular portion The transmembrane + juxtamembrane part ( a.a. + N-terminal 7His-tag) contains the transmembrane and the regulatory juxtamembrane (JM) domain

2 Important information about the tj-EGFR 73 amino acid residues (without tag) 73 amino acid residues (without tag) carries N-terminal 7His-tag carries N-terminal 7His-tag molecular weight is about 9,112 Da molecular weight is about 9,112 Da pI is around 11.5 pI is around 11.5 contains no Cys residues contains no Cys residues contains no aromatic residues (Trp, Tyr or Phe) contains no aromatic residues (Trp, Tyr or Phe) NMR structure of the juxtamembrane domain is available NMR structure of the juxtamembrane domain is available Choowongkomon et al. (2005), J. Biol. Chem.

3 Short overview of the work done 1.tj-hEGFR in DM Me-affinity Me-affinity reverse phase reverse phase cation exchange cation exchange 2.tj-hEGFR in OG Me-affinity Me-affinity reverse phase reverse phase cation exchange cation exchange size exclusion size exclusion 3.tj-hEGFR in N- lauryl sarcosine Me-affinity Me-affinity cation exchange cation exchange size exclusion size exclusion 4.tj-hEGFR in urea Me-affinity Me-affinity size exclusion size exclusion

4 Expression and solubilization of tj-EGFR in pET 27b+ in E.coli BL21(DE3) Codon Plus RP m m – marker (BenchMark) 1 – cells, clone #1 before induction 2 – cells, clone #1 after induction 3 – cell lysate, supernatant 4 – cell lysate, pellet 5 – solubilization in 2% DM, supernatant 6 – solubilization in 2% DM, pellet

5 buffer A = 50 mM NaP pH 8.0, 0.05% (w/v) DM buffer B = buffer A + 1 M NaCl Pilot purification of tj-EGFR in DM on HiTrap SP FF 1 ml m b m m – marker (BenchMark) b – sample before application

6 buffer A = 300 mM NaCl, 50 mM NaP pH 8.0, 0.05% (w/v) DM buffer B = buffer A mM imidazole also tried to use 250 mM histidine for elution: Cu 2+ ions elute at low histidine concentrations Pilot purification of tj-EGFR in DM on HiTrap Chelating 1 ml m b m – marker (BenchMark) b – sample before application

7 Scaling of purification of tj-EGFR in DM on HiTrap Chelating 1 ml m b m m m – marker (BenchMark) b – sample before application m

8 buffer A = 0.1% (w/v) TFA buffer B = 0.08% (w/v) TFA, 80% (v/v) acetonitrile Pilot purification of tj-EGFR in DM on RESOURCE RPC 3 ml m m – marker (Precision Plus)

9 buffer A = 300 mM NaCl, 50 mM NaP pH 8.0, 0.88% (w/v) OG buffer B = buffer A mM imidazole Pilot purification of tj-EGFR in OG on HiTrap Chelating 1 ml m b m – marker (BenchMark) b – sample before application

10 Scaling of purification of tj-EGFR in OG Hitrap Chelating Sepharose m S FT W1 W2 E m – marker (Precision Plus) S – sample before application (solubilized protein) FT – flow-through W1 – wash 1 W2 – wash 2 E – elution

11 Reconstitution of tj-EGFR in DMPC liposomes SDS-gelHis-blot m OG rec rec_s/_p m – marker (BenchMark) OG – sample before reconstitution (in 0.88% OG) rec – reconstituted protein rec_s – rec, supernatant rec_p – rec, pellet

12 CD spectrum of tj-EGFR and secondary structure prediction buffer = 50 mM NaP pH 6.0, 10% D 2 O, 0.88% (w/v) OG

13 buffer A = 50 mM NaP pH 8.0, 0.88% (w/v) OG buffer B = buffer A + 1 M NaCl Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 ml m b m m – marker (BenchMark) b – sample before application

14 buffer A = 50 mM NaP pH 11.7, 0.88% (w/v) OG buffer B = buffer A + 1 M NaCl Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 ml b m m – marker (BenchMark) b – sample before application

15 buffer A = 50 mM Gly-NaOH pH 8.6, 0.88% (w/v) OG buffer B = 50 mM Gly-NaOH pH 10.6, 0.88% (w/v) OG Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 ml m m m – marker (BenchMark)

16 buffer A = 50 mM NaP pH 8.0, 0.88% (w/v) OG buffer B = 50 mM NaP pH 11.5, 0.88% (w/v) OG Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 ml b m m m m – marker (BenchMark) b – sample before application

17 buffer A = 50 mM NaP pH 8.0, 0.88% (w/v) OG buffer B = buffer A + 1 M NaCl Pilot purification of tj-EGFR in OG on Mini S 4.6/50 PE m m m – marker (BenchMark)

18 buffer A = 0.1% (w/v) TFA buffer B = 0.08% (w/v) TFA, 80% (v/v) acetonitrile Pilot purification of tj-EGFR in OG on RESOURCE RPC 3 ml m m – marker (BenchMark) also tried to use TEA instead of TFA: much worse separation

19 buffer A = 20 mM NaP pH 7.0 buffer B = 70% (v/v) acetonitrile Pilot purification of tj-EGFR in OG on RESOURCE RPC 3 ml m and m – marker (BenchMark)

20 buffer A = 0.08% (w/v) TFA, 70% (v/v) acetonitrile Pilot purification of tj-EGFR in OG on Peptide Superdex 10/300 GL m m – marker (BenchMark)

21 buffer A = 150 mM NaCl, 50 mM NaP pH 8.0, 0.88% (w/v) OG Pilot purification of tj-EGFR in OG on Superdex /300 GL m m – marker (BenchMark) m

22 Pilot purification of tj-EGFR in sarcosine Hitrap Chelating Sepharose m S FT W1 W2 E m – marker (Precision Plus) S – sample before application (solubilized protein) FT – flow-through W1 – wash 1 W2 – wash 2 E – elution

23 buffer A = 50 mM NaP pH 7.0, 0.2% (w/v) N-lauryl sarcosine buffer B = buffer A + 1 M NaCl Pilot purification of tj-EGFR in sarcosine on Mini S 4.6/50 PE b m m m – marker (BenchMark) b – sample before application

24 buffer A = 300 mM NaCl, 50 mM NaP pH 8.0, 0.15% (w/v) N-lauryl sarcosine Pilot purification of tj-EGFR in sarcosine on Peptide Superdex 10/300 GL m m m – marker (BenchMark)

25 buffer A = 8 M urea, 50 mM NaP pH 8.0 buffer B = buffer A mM imidazole Pilot purification of tj-EGFR in urea on HiTrap Chelating 1 ml c m b c – crude cell extract solubilized in 8 M urea m – marker (BenchMark) b – sample before application (supernatant), in 8 M urea

26 buffer A = 8 M urea, 25 mM NaOAc pH 4.5 Pilot purification of tj-EGFR in urea on Peptide Superdex 10/300 GL m m m – marker (BenchMark)

27 buffer A = 300 mM NaCl, 50 mM NaP pH 8.0 buffer B = buffer A mM imidazole Pilot purification of tj-EGFR HiTrap Chelating 1ml m b m m m – marker (BenchMark) b – sample before application m b

28 buffer A = 50 mM NaP pH 8.0 buffer B = buffer A + 1 M NaCl Pilot purification of tj-EGFR on HiTrap SP FF 1 ml b m m m – marker (BenchMark) b – sample before application

29 What is yet to be done with tj-EGFR Compare the CD results with the new construct Compare the CD results with the new construct7His-transmembrane+juxtamembrane-StrepII Investigate dimerization (depends on lipids/detergent?!) Investigate dimerization (depends on lipids/detergent?!) Characterize in detail by CD spectroscopy under various conditions Characterize in detail by CD spectroscopy under various conditions Crystallize, do 15 N-NMR Crystallize, do 15 N-NMR

30 THANKS FOR YOUR ATTENTION