11

Epidermal Growth Factor Receptor (EGFR)Epidermal Growth Factor Receptor (EGFR)the transmembrane + juxtamembrane domainsthe transmembrane + juxtamembrane domainsEpidermal Growth Factor Receptor (EGFR)Epidermal Growth Factor Receptor (EGFR)

the transmembrane + juxtamembrane domainsthe transmembrane + juxtamembrane domains

L1 CR1 L2 CR2 JM Kinase CT

644

151 312 481 621 687 955 1186

Extracellular portion Intracellular portion

L1 CR1 L2 CR2 JM Kinase CT

644

151 312 481 621 687 955 1186

Extracellular portion Intracellular portion

The transmembrane + juxtamembrane part (615-686 a.a. + N-terminal 7His-tag) The transmembrane + juxtamembrane part (615-686 a.a. + N-terminal 7His-tag) contains the transmembrane and the regulatory juxtamembrane (JM) domaincontains the transmembrane and the regulatory juxtamembrane (JM) domain

22

Important information about the tj-EGFRImportant information about the tj-EGFRImportant information about the tj-EGFRImportant information about the tj-EGFR

73 amino acid residues (without tag)73 amino acid residues (without tag) carries N-terminal 7His-tagcarries N-terminal 7His-tag molecular weight is about 9,112 Damolecular weight is about 9,112 Da pI is around 11.5pI is around 11.5 contains no Cys residuescontains 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 availableNMR structure of the juxtamembrane domain is availableChoowongkomonChoowongkomon et alet al. (2005), J. Biol. Chem.. (2005), J. Biol. Chem.

33

Short overview of the work doneShort overview of the work doneShort overview of the work doneShort overview of the work done

1.1. tj-hEGFR in DMtj-hEGFR in DM

Me-affinityMe-affinity reverse phasereverse phase cation exchangecation exchange

2.2. tj-hEGFR in OGtj-hEGFR in OG

Me-affinityMe-affinity reverse phasereverse phase cation exchangecation exchange size exclusionsize exclusion

3.3. tj-hEGFR in N-tj-hEGFR in N-lauryl sarcosinelauryl sarcosine

Me-affinityMe-affinity cation exchangecation exchange size exclusionsize exclusion

4.4. tj-hEGFR in ureatj-hEGFR in urea

Me-affinityMe-affinity size exclusionsize exclusion

44

Expression and solubilization of tj-EGFR in pET Expression and solubilization of tj-EGFR in pET 27b+ in 27b+ in E.coliE.coli BL21(DE3) Codon Plus RP BL21(DE3) Codon Plus RP

Expression and solubilization of tj-EGFR in pET Expression and solubilization of tj-EGFR in pET 27b+ in 27b+ in E.coliE.coli BL21(DE3) Codon Plus RP BL21(DE3) Codon Plus RP

m 1 2 3 4 5 6m 1 2 3 4 5 6

m – marker (BenchMark)m – marker (BenchMark)1 – cells, clone #1 before induction1 – cells, clone #1 before induction2 – cells, clone #1 after induction2 – cells, clone #1 after induction3 – cell lysate, supernatant3 – cell lysate, supernatant4 – cell lysate, pellet4 – cell lysate, pellet

5 – solubilization in 2% DM, supernatant5 – solubilization in 2% DM, supernatant6 – solubilization in 2% DM, pellet6 – solubilization in 2% DM, pellet

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buffer A = 50 mM NaP pH 8.0, 0.05% (w/v) DMbuffer A = 50 mM NaP pH 8.0, 0.05% (w/v) DMbuffer B = buffer A + 1 M NaClbuffer B = buffer A + 1 M NaCl

Pilot purification of tj-EGFR in DM on Pilot purification of tj-EGFR in DM on HiTrap SP FF 1 mlHiTrap SP FF 1 ml

Pilot purification of tj-EGFR in DM on Pilot purification of tj-EGFR in DM on HiTrap SP FF 1 mlHiTrap SP FF 1 ml

m b 1 2 3 4 5 6 7 8m b 1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 m9 11 12 13 14 15 16 17 18 m

m – marker (BenchMark)m – marker (BenchMark)b – sample before applicationb – sample before application

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66

buffer A = 300 mM NaCl, 50 mM NaP pH 8.0,buffer A = 300 mM NaCl, 50 mM NaP pH 8.0,0.05% (w/v) DM0.05% (w/v) DMbuffer B = buffer A + 500 mM imidazolebuffer B = buffer A + 500 mM imidazole

also tried to use 250 mM histidine for elution:also tried to use 250 mM histidine for elution:CuCu2+2+ ions elute at low histidine concentrations ions elute at low histidine concentrations

Pilot purification of tj-EGFR in DM on Pilot purification of tj-EGFR in DM on HiTrap Chelating 1 mlHiTrap Chelating 1 ml

Pilot purification of tj-EGFR in DM on Pilot purification of tj-EGFR in DM on HiTrap Chelating 1 mlHiTrap Chelating 1 ml

m b 2 9 10 11 13 15 17 19m b 2 9 10 11 13 15 17 19

m – marker (BenchMark)m – marker (BenchMark)b – sample before applicationb – sample before application

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77

Scaling of purification of tj-EGFR in DM on Scaling of purification of tj-EGFR in DM on HiTrap Chelating 1 mlHiTrap Chelating 1 ml

Scaling of purification of tj-EGFR in DM on Scaling of purification of tj-EGFR in DM on HiTrap Chelating 1 mlHiTrap Chelating 1 ml

m b 1 2 3 4 5 6 7 8m b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 17 19 9 10 11 12 13 14 15 17 19 mm

m – marker (BenchMark)m – marker (BenchMark)b – sample before applicationb – sample before applicationm 14 16 17 18 20 22m 14 16 17 18 20 22

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88

buffer A = 0.1% (w/v) TFAbuffer A = 0.1% (w/v) TFAbuffer B = 0.08% (w/v) TFA, 80% (v/v) acetonitrilebuffer B = 0.08% (w/v) TFA, 80% (v/v) acetonitrile

Pilot purification of tj-EGFR in DM on Pilot purification of tj-EGFR in DM on RESOURCE RPC 3 mlRESOURCE RPC 3 ml

Pilot purification of tj-EGFR in DM on Pilot purification of tj-EGFR in DM on RESOURCE RPC 3 mlRESOURCE RPC 3 ml

m 2 4 7 8 12 13 14 17 18m 2 4 7 8 12 13 14 17 18

m – marker (Precision Plus)m – marker (Precision Plus)

2020

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99

buffer A = 300 mM NaCl, 50 mM NaP pH 8.0,buffer A = 300 mM NaCl, 50 mM NaP pH 8.0,0.88% (w/v) OG0.88% (w/v) OGbuffer B = buffer A + 500 mM imidazolebuffer B = buffer A + 500 mM imidazole

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on HiTrap Chelating 1 mlHiTrap Chelating 1 ml

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on HiTrap Chelating 1 mlHiTrap Chelating 1 ml

m b 2 8 9 10 12 14 16 18m b 2 8 9 10 12 14 16 18

m – marker (BenchMark)m – marker (BenchMark)b – sample before applicationb – sample before application

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1010

Scaling of purification of tj-EGFR in OG Scaling of purification of tj-EGFR in OG Hitrap Chelating SepharoseHitrap Chelating Sepharose

Scaling of purification of tj-EGFR in OG Scaling of purification of tj-EGFR in OG Hitrap Chelating SepharoseHitrap Chelating Sepharose

m S FT W1 W2 Em S FT W1 W2 E

m – marker (Precision Plus)m – marker (Precision Plus)S – sample before application S – sample before application (solubilized protein)(solubilized protein)FT – flow-throughFT – flow-throughW1 – wash 1W1 – wash 1W2 – wash 2W2 – wash 2E – elution E – elution

2020

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Reconstitution of tj-EGFR in DMPC liposomesReconstitution of tj-EGFR in DMPC liposomesReconstitution of tj-EGFR in DMPC liposomesReconstitution of tj-EGFR in DMPC liposomes

SDS-gelSDS-gel His-blotHis-blot

m OG rec rec_s/_pm OG rec rec_s/_p m OG rec rec_s/_pm OG rec rec_s/_p

m – marker (BenchMark)m – marker (BenchMark)OG – sample before OG – sample before reconstitution (in 0.88% OG)reconstitution (in 0.88% OG)rec – reconstituted proteinrec – reconstituted proteinrec_s – rec, supernatantrec_s – rec, supernatantrec_p – rec, pelletrec_p – rec, pellet

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1212

CD spectrum of tj-EGFR and CD spectrum of tj-EGFR and secondary structure predictionsecondary structure predictionCD spectrum of tj-EGFR and CD spectrum of tj-EGFR and secondary structure predictionsecondary structure prediction

buffer = 50 mM NaP pH 6.0, 10% Dbuffer = 50 mM NaP pH 6.0, 10% D22O, 0.88% (w/v) OGO, 0.88% (w/v) OG

1313

buffer A = 50 mM NaP pH 8.0, buffer A = 50 mM NaP pH 8.0, 0.88% (w/v) OG0.88% (w/v) OGbuffer B = buffer A + 1 M NaClbuffer B = buffer A + 1 M NaCl

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 mlHiTrap SP FF 1 ml

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 mlHiTrap SP FF 1 ml

m b 2 4 5 6 9 10 11 12m b 2 4 5 6 9 10 11 12 13 14 15 16 17 18 19 20 21 m13 14 15 16 17 18 19 20 21 m

m – marker (BenchMark)m – marker (BenchMark)b – sample before applicationb – sample before application

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1414

buffer A = 50 mM NaP pH 11.7, buffer A = 50 mM NaP pH 11.7, 0.88% (w/v) OG0.88% (w/v) OGbuffer B = buffer A + 1 M NaClbuffer B = buffer A + 1 M NaCl

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 mlHiTrap SP FF 1 ml

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 mlHiTrap SP FF 1 ml

b m 2 3 8 11 12 13 15 16b m 2 3 8 11 12 13 15 16

m – marker (BenchMark)m – marker (BenchMark)b – sample before applicationb – sample before application

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buffer A = 50 mM Gly-NaOH pH 8.6, buffer A = 50 mM Gly-NaOH pH 8.6, 0.88% (w/v) OG0.88% (w/v) OGbuffer B = 50 mM Gly-NaOH pH 10.6, buffer B = 50 mM Gly-NaOH pH 10.6, 0.88% (w/v) OG0.88% (w/v) OG

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 mlHiTrap SP FF 1 ml

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 mlHiTrap SP FF 1 ml

2 4 6 8 m 19 20 21 22 23 24 25 26 27 282 4 6 8 m 19 20 21 22 23 24 25 26 27 28 m 29 30 31 32 33 34 35 36 37 38 39 40 41 42m 29 30 31 32 33 34 35 36 37 38 39 40 41 42

m – marker (BenchMark)m – marker (BenchMark)

1616

buffer A = 50 mM NaP pH 8.0, buffer A = 50 mM NaP pH 8.0, 0.88% (w/v) OG0.88% (w/v) OGbuffer B = 50 mM NaP pH 11.5, buffer B = 50 mM NaP pH 11.5, 0.88% (w/v) OG0.88% (w/v) OG

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 mlHiTrap SP FF 1 ml

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 mlHiTrap SP FF 1 ml

b m 2 4 8 14 16 18 20 22b m 2 4 8 14 16 18 20 22 24 26 28 30 32 34 36 38 40 m24 26 28 30 32 34 36 38 40 m

m – marker (BenchMark)m – marker (BenchMark)b – sample before applicationb – sample before application

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buffer A = 50 mM NaP pH 8.0, buffer A = 50 mM NaP pH 8.0, 0.88% (w/v) OG0.88% (w/v) OGbuffer B = buffer A + 1 M NaClbuffer B = buffer A + 1 M NaCl

Pilot purification of tj-EGFR in OG onPilot purification of tj-EGFR in OG onMini S 4.6/50 PEMini S 4.6/50 PE

Pilot purification of tj-EGFR in OG onPilot purification of tj-EGFR in OG onMini S 4.6/50 PEMini S 4.6/50 PE

m 3 8 25 30 31 32 33 34 35m 3 8 25 30 31 32 33 34 35 37 39 41 43 45 47 49 51 52 m37 39 41 43 45 47 49 51 52 m

m – marker (BenchMark)m – marker (BenchMark)

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buffer A = 0.1% (w/v) TFAbuffer A = 0.1% (w/v) TFAbuffer B = 0.08% (w/v) TFA, 80% (v/v) acetonitrilebuffer B = 0.08% (w/v) TFA, 80% (v/v) acetonitrile

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on RESOURCE RPC 3 mlRESOURCE RPC 3 ml

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on RESOURCE RPC 3 mlRESOURCE RPC 3 ml

m 7 12 24 33 35 38 39 40 42m 7 12 24 33 35 38 39 40 42

m – marker (BenchMark)m – marker (BenchMark)

also tried to use TEA instead of TFA:also tried to use TEA instead of TFA:much worse separationmuch worse separation

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buffer A = 20 mM NaP pH 7.0buffer A = 20 mM NaP pH 7.0buffer B = 70% (v/v) acetonitrilebuffer B = 70% (v/v) acetonitrile

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on RESOURCE RPC 3 mlRESOURCE RPC 3 ml

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on RESOURCE RPC 3 mlRESOURCE RPC 3 ml

m 10 11 13 15 16 and 22-35m 10 11 13 15 16 and 22-35

m – marker (BenchMark)m – marker (BenchMark)

2020

buffer A = 0.08% (w/v) TFA, 70% (v/v) acetonitrilebuffer A = 0.08% (w/v) TFA, 70% (v/v) acetonitrile

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on Peptide Superdex 10/300 GLPeptide Superdex 10/300 GL

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on Peptide Superdex 10/300 GLPeptide Superdex 10/300 GL

m 11 12 13 14 15 16 17 22 23m 11 12 13 14 15 16 17 22 23

m – marker (BenchMark)m – marker (BenchMark)

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2121

buffer A = 150 mM NaCl,buffer A = 150 mM NaCl,50 mM NaP pH 8.0,50 mM NaP pH 8.0,0.88% (w/v) OG0.88% (w/v) OG

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on Superdex 200 10/300 GLSuperdex 200 10/300 GL

Pilot purification of tj-EGFR in OG on Pilot purification of tj-EGFR in OG on Superdex 200 10/300 GLSuperdex 200 10/300 GL

m 5 8 9 10 11 12 13 14 15m 5 8 9 10 11 12 13 14 15

m – marker (BenchMark)m – marker (BenchMark)

18 19 20 21 22 23 24 25 26 m18 19 20 21 22 23 24 25 26 m

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Pilot purification of tj-EGFR in sarcosine Pilot purification of tj-EGFR in sarcosine Hitrap Chelating SepharoseHitrap Chelating Sepharose

Pilot purification of tj-EGFR in sarcosine Pilot purification of tj-EGFR in sarcosine Hitrap Chelating SepharoseHitrap Chelating Sepharose

m S FT W1 W2 Em S FT W1 W2 E

m – marker (Precision Plus)m – marker (Precision Plus)S – sample before application S – sample before application (solubilized protein)(solubilized protein)FT – flow-throughFT – flow-throughW1 – wash 1W1 – wash 1W2 – wash 2W2 – wash 2E – elution E – elution

2020

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2323

buffer A = 50 mM NaP pH 7.0, buffer A = 50 mM NaP pH 7.0, 0.2% (w/v) N-lauryl sarcosine0.2% (w/v) N-lauryl sarcosinebuffer B = buffer A + 1 M NaClbuffer B = buffer A + 1 M NaCl

Pilot purification of tj-EGFR in sarcosine Pilot purification of tj-EGFR in sarcosine on Mini S 4.6/50 PEon Mini S 4.6/50 PE

Pilot purification of tj-EGFR in sarcosine Pilot purification of tj-EGFR in sarcosine on Mini S 4.6/50 PEon Mini S 4.6/50 PE

b m 3 5 13 15 17 19 21 b m 3 5 13 15 17 19 21 2323

25 27 29 31 33 35 37 39 41 m25 27 29 31 33 35 37 39 41 m

m – marker (BenchMark)m – marker (BenchMark)b – sample before applicationb – sample before application

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buffer A = 300 mM NaCl, 50 mM NaP buffer A = 300 mM NaCl, 50 mM NaP pH 8.0, 0.15% (w/v) N-lauryl sarcosinepH 8.0, 0.15% (w/v) N-lauryl sarcosine

Pilot purification of tj-EGFR in sarcosine on Pilot purification of tj-EGFR in sarcosine on Peptide Superdex 10/300 GLPeptide Superdex 10/300 GL

Pilot purification of tj-EGFR in sarcosine on Pilot purification of tj-EGFR in sarcosine on Peptide Superdex 10/300 GLPeptide Superdex 10/300 GL

m 17 18 19 20 21 22 23 24 25m 17 18 19 20 21 22 23 24 25

m – marker (BenchMark)m – marker (BenchMark)

2525

buffer A = 8 M urea, 50 mM NaP pH 8.0buffer A = 8 M urea, 50 mM NaP pH 8.0buffer B = buffer A + 500 mM imidazolebuffer B = buffer A + 500 mM imidazole

Pilot purification of tj-EGFR in urea on Pilot purification of tj-EGFR in urea on HiTrap Chelating 1 mlHiTrap Chelating 1 ml

Pilot purification of tj-EGFR in urea on Pilot purification of tj-EGFR in urea on HiTrap Chelating 1 mlHiTrap Chelating 1 ml

c m b 2 10 12 14 16 18 20c m b 2 10 12 14 16 18 20

c – crude cell extract solubilized in 8 M ureac – crude cell extract solubilized in 8 M uream – marker (BenchMark)m – marker (BenchMark)b – sample before application (supernatant), in 8 M ureab – sample before application (supernatant), in 8 M urea

2626

buffer A = 8 M urea, 25 mM NaOAc pH 4.5buffer A = 8 M urea, 25 mM NaOAc pH 4.5

Pilot purification of tj-EGFR in urea on Pilot purification of tj-EGFR in urea on Peptide Superdex 10/300 GLPeptide Superdex 10/300 GL

Pilot purification of tj-EGFR in urea on Pilot purification of tj-EGFR in urea on Peptide Superdex 10/300 GLPeptide Superdex 10/300 GL

m 5 13 14 15 16 17 18 19 20m 5 13 14 15 16 17 18 19 20

m – marker (BenchMark)m – marker (BenchMark)

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buffer A = 300 mM NaCl, 50 mM NaP pH 8.0buffer A = 300 mM NaCl, 50 mM NaP pH 8.0buffer B = buffer A + 500 mM imidazolebuffer B = buffer A + 500 mM imidazole

Pilot purification of tj-EGFR HiTrap Chelating 1mlPilot purification of tj-EGFR HiTrap Chelating 1mlPilot purification of tj-EGFR HiTrap Chelating 1mlPilot purification of tj-EGFR HiTrap Chelating 1ml

m b 1 3 10 11 12 13 14 15m b 1 3 10 11 12 13 14 15 16 18 20 22 24 25 26 27 29 m16 18 20 22 24 25 26 27 29 m

m – marker (BenchMark)m – marker (BenchMark)b – sample before applicationb – sample before application

m b 1 11 12 13 14 15 16 18m b 1 11 12 13 14 15 16 18

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buffer A = 50 mM NaP pH 8.0buffer A = 50 mM NaP pH 8.0buffer B = buffer A + 1 M NaClbuffer B = buffer A + 1 M NaCl

Pilot purification of tj-EGFR on HiTrap SP FF 1 mlPilot purification of tj-EGFR on HiTrap SP FF 1 mlPilot purification of tj-EGFR on HiTrap SP FF 1 mlPilot purification of tj-EGFR on HiTrap SP FF 1 ml

b m 2 4 15 16 17 19 20 21b m 2 4 15 16 17 19 20 21 23 25 27 29 31 32 33 34 35 m23 25 27 29 31 32 33 34 35 m

m – marker (BenchMark)m – marker (BenchMark)b – sample before applicationb – sample before application

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What is yet to be done with tj-EGFRWhat is yet to be done with tj-EGFRWhat is yet to be done with tj-EGFRWhat is yet to be done with tj-EGFR

Compare the CD results with the new constructCompare the CD results with the new construct

7His-transmembrane+juxtamembrane-StrepII7His-transmembrane+juxtamembrane-StrepII

Investigate dimerization (depends on Investigate dimerization (depends on lipids/detergent?!)lipids/detergent?!)

Characterize in detail by CD spectroscopy under Characterize in detail by CD spectroscopy under various conditionsvarious conditions

Crystallize, do Crystallize, do 1515N-NMRN-NMR

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THANKS FOR YOUR ATTENTIONTHANKS FOR YOUR ATTENTIONTHANKS FOR YOUR ATTENTIONTHANKS FOR YOUR ATTENTION