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Rhodopsin: Effect of replacement of amino acids in the predicted folding core of Rhodopsin

Rhodopsin: Effect of replacement of amino acids in the predicted folding core of Rhodopsin Growth hormone receptor (GHR): Expression of Cytoplasmic domain of GHR4 in E.coli, Epidermal Growth Factor (EGF): Secretory and extracelluar production of human EGF

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Rhodopsin: Effect of replacement of amino acids in the predicted folding core of Rhodopsin

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  1. Rhodopsin: Effect of replacement of amino acids in the predicted folding core of Rhodopsin • Growth hormone receptor (GHR): Expression of Cytoplasmic domain of GHR4 in E.coli, • Epidermal Growth Factor (EGF): Secretory and extracelluar production of human EGF • Ciliary Neurotrophic Factor Alpha Receptor (CNTFR): Expression of Cytokine binding domain (CBD) of CNTFR

  2. Rhodopsin

  3. A166 F116 A168 A169 L112 N111 F24 A26 P27 S22 E25

  4. Bovine Rhodopsin GAATTCATGAACGGTACCGAAGGCCCAAACTTCTACGTTCCTTTCTCCAACAAGACGGGCGTGGTGCGCAGCCCGTTCGAGGCTCCGCAGTACTACCTGGCGGAGCCCTGGCAGTTCTCCATGCTGGCCGCCTACATGTTCCTGCTGATCATGCTTGGCTTCCCGATCAACTTCCTCACGCTGTACGTCACAGTCCAGCACAAGAAGCTTCGCACACCGCTCAACTACATCCTGCTCAACCTGGCCGTGGCAGATCTCTTCATGGTCTTCGGTGGCTTCACCACCACCCTCTACACCTCTCTCCATGGGTACTTCGTCTTTGGGCCGACGGGCTGCAACCTCGAGGGCTTCTTTGCCACCCTGGGCGGTGAAATTGCACTGTGGTCTCTGGTAGTACTGGCGATCGAGCGGTACGTGGTGGTGTGCAAGCCCATGAGCAACTTCCGCTTCGGTGAGAACCACGCCATCATGGGCGTCGCCTTCACCTGGGTCATGGCTCTGGCCTGTGCGGCCCCGCCGCTCGTCGGCTGGTCTAGATACATCCCGGAGGGCATGCAGTGCTCGTGCGGGATCGATTACTACACGCCGCACGAGGAGACCAACAATGAGTCGTTCGTCATCTACATGTTCGTGGTCCACTTCATCATCCCGCTGATTGTCATCTTCTTCTGCTATGGCCAGCTGGTGTTCACCGTCAAGGAGGCTGCAGCCCAGCAGCAGGAGAGCGCCACCACTCAGAAGGCCGAGAAGGAGGTCACGCGTATGGTTATCATCATGGTCATCGCTTTCCTAATCTGCTGGCTGCCCTATGCTGGTGTGGCGTTCTACATCTTCACCCATCAGGGCTCTGACTTTGGGCCCATCTTCATGACCATCCCGGCTTTCTTTGCCAAGACGTCTGCCGTCTACAACCCGGTCATCTACATCATGATGAACAAGCAGTTCCGGAACTGCATGGTCACCACTCTCTGCTGTGGCAAGAACCCGCTGGGTGACGACGAGGCGTCGACCACCGTCTCCAAGACAGAGACCAGCCAAGTGGCGCCTGCCTAAGCGGCCGC MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIMLGFPINFLTLYVTVQHKKLRTPLNYILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFGENHAIMGVAFTWVMALACAAPPLVGWSRYIPEGMQCSCGIDYYTPHEETNNESFVIYMFVVHFIIPLIVIFFCYGQLVFTVKEAAAQQQESATTQKAEKEVTRMVIIMVIAFLICWLPYAGVAFYIFTHQGSDFGPIFMTIPAFFAKTSAVYNPVIYIMMNKQFRNCMVTTLCCGKNPLGDDEASTTVSKTETSQVAPA (Underlined nucleotide has been modified, opsin gene in PMT4 is different to the original sequence as shown above)

  5. Relative ratio A280/A500 of rhodopsin wide Type (WT) and 11 mutants Mutants F24C, A26C, L112C/F116C, F116C and A168C showed relative ratio between 500nm and 280nm were greater than 8 Mutants S22C, E25C, A27C, N111C, A166C and A169C showed less than 5 [Further experiments are still need to perform to further verify before the classified the mutants]

  6. Elution profile of Rhodopsin mutant A169C first with NaPi buffer and then followed buffer with 150mM NaCl

  7. Rhodopsin was labeled with 50uM NEM in 50uM NaPi, 0.05%DM in dark for at least 16 hrs Wash with 5mM MES, 0.05%DM 50uM spin label MTSL was added for 3 hrs RT Rhodopsin was labeled with 50uM NEM in 20mM Tris, 0.05%DM, 150mM NaCl in dark for at least 16 hrs Wash with 20mM Tris, 0.05%DM, 150mM NaCl 50uM spin label MTSL was added overnight at 4C Spin labeling experiment for electron paramagnetic resonance (EPR) spectroscopy • R9 and R11 • After extensively washing with 1XPBS, 0.05%DM followed by 2mM NaPi, 0.05% DM of solubilized Rhodopsin-1D4 Sepharose • Washing 5mM MES, 0.05%DM • Labeled Rhodopsin were eluted 70uM epitope nonapeptide

  8. Epidermal Growth Factor

  9. Purification of hEGF through the ion-exchange column Control Sample kDa 75 50 37 25 hEGF

  10. Ciliary Neurotrophic Factor Alpha Receptor

  11. kDa 75 50 37 25 M Elute Sol On-column digestion MBP-CBD MBP CBD

  12. ACKNOWLEDGMENTS Judith Klein-Seetharaman Thanks To those prepare to work long hours with little materialistic reward, misunderstood by most people, take part (order around) in teamwork as well as slave along during the lonely nights merely to solve a few interesting problems in biology......

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