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Epidermal Growth Factor Receptor (EGFR) the transmembrane + juxtamembrane domains

151. 151. 312. 312. 481. 481. 621. 621. 687. 687. 955. 955. 1186. 1186. L1. L1. CR1. CR1. L2. L2. CR2. CR2. Kinase. Kinase. CT. CT. JM. JM. 644. 644. Extracellular portion. Extracellular portion. Intracellular portion. Intracellular portion.

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Epidermal Growth Factor Receptor (EGFR) the transmembrane + juxtamembrane domains

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  1. 151 151 312 312 481 481 621 621 687 687 955 955 1186 1186 L1 L1 CR1 CR1 L2 L2 CR2 CR2 Kinase Kinase CT CT JM JM 644 644 Extracellular portion Extracellular portion Intracellular portion Intracellular portion Epidermal Growth Factor Receptor (EGFR)the transmembrane + juxtamembrane domains The transmembrane + juxtamembrane part (615-686 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) • carries N-terminal 7His-tag • molecular weight is about 9,112 Da • pI is around 11.5 • contains no Cys residues • contains no aromatic residues (Trp, Tyr or Phe) • NMR structure of the juxtamembrane domain is available Choowongkomonet al. (2005), J. Biol. Chem.

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

  4. 20 15 10 Expression and solubilization of tj-EGFR in pET 27b+ in E.coli BL21(DE3) Codon Plus RP m 1 2 3 4 5 6 m – marker (BenchMark)1 – cells, clone #1 before induction2 – cells, clone #1 after induction3 – cell lysate, supernatant4 – cell lysate, pellet 5 – solubilization in 2% DM, supernatant6 – solubilization in 2% DM, pellet

  5. 20 20 15 15 10 10 Pilot purification of tj-EGFR in DM on HiTrap SP FF 1 ml buffer A = 50 mM NaP pH 8.0, 0.05% (w/v) DMbuffer B = buffer A + 1 M NaCl m b 1 2 3 4 5 6 7 8 9 11 12 13 14 15 16 17 18 m m – marker (BenchMark)b – sample before application

  6. 20 15 10 Pilot purification of tj-EGFR in DM on HiTrap Chelating 1 ml buffer A = 300 mM NaCl, 50 mM NaP pH 8.0,0.05% (w/v) DMbuffer B = buffer A + 500 mM imidazolealso tried to use 250 mM histidine for elution:Cu2+ ions elute at low histidine concentrations m b 2 9 10 11 13 15 17 19 m – marker (BenchMark)b – sample before application

  7. 20 20 15 15 25 10 10 20 15 Scaling of purification of tj-EGFR in DM on HiTrap Chelating 1 ml m b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 17 19 m m – marker (BenchMark)b – sample before application m 14 16 17 18 20 22

  8. Pilot purification of tj-EGFR in DM on RESOURCE RPC 3 ml buffer A = 0.1% (w/v) TFAbuffer B = 0.08% (w/v) TFA, 80% (v/v) acetonitrile m 2 4 7 8 12 13 14 17 18 25 20 15 m – marker (Precision Plus)

  9. 20 15 10 Pilot purification of tj-EGFR in OG on HiTrap Chelating 1 ml buffer A = 300 mM NaCl, 50 mM NaP pH 8.0,0.88% (w/v) OGbuffer B = buffer A + 500 mM imidazole m b 2 8 9 10 12 14 16 18 m – marker (BenchMark)b – sample before application

  10. 25 20 15 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-throughW1 – wash 1W2 – wash 2E – elution

  11. 20 20 15 15 10 10 Reconstitution of tj-EGFR in DMPC liposomes m OG rec rec_s/_p m OG rec rec_s/_p m – marker (BenchMark)OG – sample before reconstitution (in 0.88% OG)rec – reconstituted proteinrec_s – rec, supernatantrec_p – rec, pellet SDS-gel His-blot

  12. CD spectrum of tj-EGFR and secondary structure prediction buffer = 50 mM NaP pH 6.0, 10% D2O, 0.88% (w/v) OG

  13. 20 20 15 15 10 10 Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 ml buffer A = 50 mM NaP pH 8.0, 0.88% (w/v) OGbuffer B = buffer A + 1 M NaCl m b 2 4 5 6 9 10 11 12 13 14 15 16 17 18 19 20 21 m m – marker (BenchMark)b – sample before application

  14. 20 15 10 Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 ml buffer A = 50 mM NaP pH 11.7, 0.88% (w/v) OGbuffer B = buffer A + 1 M NaCl b m 2 3 8 11 12 13 15 16 m – marker (BenchMark)b – sample before application

  15. Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 ml buffer A = 50 mM Gly-NaOH pH 8.6, 0.88% (w/v) OGbuffer B = 50 mM Gly-NaOH pH 10.6, 0.88% (w/v) OG 2 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 42 m – marker (BenchMark)

  16. 20 20 15 15 10 10 Pilot purification of tj-EGFR in OG on HiTrap SP FF 1 ml buffer A = 50 mM NaP pH 8.0, 0.88% (w/v) OGbuffer B = 50 mM NaP pH 11.5, 0.88% (w/v) OG b m 2 4 8 14 16 18 20 22 24 26 28 30 32 34 36 38 40 m m – marker (BenchMark)b – sample before application

  17. 20 20 15 15 10 10 Pilot purification of tj-EGFR in OG onMini S 4.6/50 PE buffer A = 50 mM NaP pH 8.0, 0.88% (w/v) OGbuffer B = buffer A + 1 M NaCl m 3 8 25 30 31 32 33 34 35 37 39 41 43 45 47 49 51 52 m m – marker (BenchMark)

  18. 20 15 10 Pilot purification of tj-EGFR in OG on RESOURCE RPC 3 ml buffer A = 0.1% (w/v) TFAbuffer B = 0.08% (w/v) TFA, 80% (v/v) acetonitrile also tried to use TEA instead of TFA:much worse separation m 7 12 24 33 35 38 39 40 42 m – marker (BenchMark)

  19. Pilot purification of tj-EGFR in OG on RESOURCE RPC 3 ml buffer A = 20 mM NaP pH 7.0buffer B = 70% (v/v) acetonitrile m 10 11 13 15 16 and 22-35 m – marker (BenchMark)

  20. Pilot purification of tj-EGFR in OG on Peptide Superdex 10/300 GL buffer A = 0.08% (w/v) TFA, 70% (v/v) acetonitrile m 11 12 13 14 15 16 17 22 23 20 15 10 m – marker (BenchMark)

  21. 20 20 15 15 10 10 Pilot purification of tj-EGFR in OG on Superdex 200 10/300 GL buffer A = 150 mM NaCl,50 mM NaP pH 8.0,0.88% (w/v) OG m 5 8 9 10 11 12 13 14 15 18 19 20 21 22 23 24 25 26 m m – marker (BenchMark)

  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-throughW1 – wash 1W2 – wash 2E – elution 25 20 15 10

  23. 20 20 15 15 10 10 Pilot purification of tj-EGFR in sarcosine on Mini S 4.6/50 PE buffer A = 50 mM NaP pH 7.0, 0.2% (w/v) N-lauryl sarcosinebuffer B = buffer A + 1 M NaCl b m 3 5 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 m m – marker (BenchMark)b – sample before application

  24. Pilot purification of tj-EGFR in sarcosine on Peptide Superdex 10/300 GL buffer A = 300 mM NaCl, 50 mM NaP pH 8.0, 0.15% (w/v) N-lauryl sarcosine m 17 18 19 20 21 22 23 24 25 m – marker (BenchMark)

  25. Pilot purification of tj-EGFR in urea on HiTrap Chelating 1 ml buffer A = 8 M urea, 50 mM NaP pH 8.0buffer B = buffer A + 500 mM imidazole c m b 2 10 12 14 16 18 20 c – crude cell extract solubilized in 8 M uream – marker (BenchMark)b – sample before application (supernatant), in 8 M urea

  26. 20 15 10 Pilot purification of tj-EGFR in urea on Peptide Superdex 10/300 GL buffer A = 8 M urea, 25 mM NaOAc pH 4.5 m 5 13 14 15 16 17 18 19 20 m – marker (BenchMark)

  27. 20 20 15 15 10 10 Pilot purification of tj-EGFR HiTrap Chelating 1ml buffer A = 300 mM NaCl, 50 mM NaP pH 8.0buffer B = buffer A + 500 mM imidazole m b 1 11 12 13 14 15 16 18 m b 1 3 10 11 12 13 14 15 16 18 20 22 24 25 26 27 29 m m – marker (BenchMark)b – sample before application

  28. 20 20 15 15 10 10 Pilot purification of tj-EGFR on HiTrap SP FF 1 ml buffer A = 50 mM NaP pH 8.0buffer B = buffer A + 1 M NaCl b m 2 4 15 16 17 19 20 21 23 25 27 29 31 32 33 34 35 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 7His-transmembrane+juxtamembrane-StrepII • Investigate dimerization (depends on lipids/detergent?!) • Characterize in detail by CD spectroscopy under various conditions • Crystallize, do 15N-NMR

  30. THANKS FOR YOUR ATTENTION

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