1 Rhodopsin: Effect of replacement of amino acids in the predicted folding core of Rhodopsin Growth hormone receptor (GHR): Expression of Cytoplasmic domain.

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Presentation transcript:

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

4 ResidueName S22CR1 F24CR2 E25CR3 A26CR4 A27CR5 N111CR6 L112C/F116CdR7 L112CR7 F116CR8 A166CR9 A168CR10 A169CR11 F116 L112 F24 S22 E25 P27 A26 A168 A166 A169 N111

5 Bovine Rhodopsin GAATTCATGAACGGTACCGAAGGCCCAAACTTCTACGTTCCTTTCTCCAACAAGACGGGCGTGGTGCGCA GCCCGTTCGAGGCTCCGCAGTACTACCTGGCGGAGCCCTGGCAGTTCTCCATGCTGGCCGCCTACATGTTC CTGCTGATCATGCTTGGCTTCCCGATCAACTTCCTCACGCTGTACGTCACAGTCCAGCACAAGAAGCTTCGC ACACCGCTCAACTACATCCTGCTCAACCTGGCCGTGGCAGATCTCTTCATGGTCTTCGGTGGCTTCACCAC CACCCTCTACACCTCTCTCCATGGGTACTTCGTCTTTGGGCCGACGGGCTGCAACCTCGAGGGCTTCTTTG CCACCCTGGGCGGTGAAATTGCACTGTGGTCTCTGGTAGTACTGGCGATCGAGCGGTACGTGGTGGTGTGC AAGCCCATGAGCAACTTCCGCTTCGGTGAGAACCACGCCATCATGGGCGTCGCCTTCACCTGGGTCATGGC TCTGGCCTGTGCGGCCCCGCCGCTCGTCGGCTGGTCTAGATACATCCCGGAGGGCATGCAGTGCTCGTGCG GGATCGATTACTACACGCCGCACGAGGAGACCAACAATGAGTCGTTCGTCATCTACATGTTCGTGGTCCAC TTCATCATCCCGCTGATTGTCATCTTCTTCTGCTATGGCCAGCTGGTGTTCACCGTCAAGGAGGCTGCAGCC CAGCAGCAGGAGAGCGCCACCACTCAGAAGGCCGAGAAGGAGGTCACGCGTATGGTTATCATCATGGTCA TCGCTTTCCTAATCTGCTGGCTGCCCTATGCTGGTGTGGCGTTCTACATCTTCACCCATCAGGGCTCTGACTT TGGGCCCATCTTCATGACCATCCCGGCTTTCTTTGCCAAGACGTCTGCCGTCTACAACCCGGTCATCTACAT CATGATGAACAAGCAGTTCCGGAACTGCATGGTCACCACTCTCTGCTGTGGCAAGAACCCGCTGGGTGACG ACGAGGCGTCGACCACCGTCTCCAAGACAGAGACCAGCCAAGTGGCGCCTGCCTAAGCGGCCGC MNGTEGPNFYVPFSNKTGVVRSPFEAPQYYLAEPWQFSMLAAYMFLLIMLGFPINFLTLYVTVQHKKLRTPLNY ILLNLAVADLFMVFGGFTTTLYTSLHGYFVFGPTGCNLEGFFATLGGEIALWSLVVLAIERYVVVCKPMSNFRFG ENHAIMGVAFTWVMALACAAPPLVGWSRYIPEGMQCSCGIDYYTPHEETNNESFVIYMFVVHFIIPLIVIFFCYG QLVFTVKEAAAQQQESATTQKAEKEVTRMVIIMVIAFLICWLPYAGVAFYIFTHQGSDFGPIFMTIPAFFAKTSAV YNPVIYIMMNKQFRNCMVTTLCCGKNPLGDDEASTTVSKTETSQVAPA (Underlined nucleotide has been modified, opsin gene in PMT4 is different to the original sequence as shown above)

6

7 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]

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

9 Spin labeling experiment for electron paramagnetic resonance (EPR) spectroscopy 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 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

10

11 Epidermal Growth Factor

12 hEGF Control Sample kDa Purification of hEGF through the ion-exchange column

13 Ciliary Neurotrophic Factor Alpha Receptor

14

15 kDa M Elute Sol On-column digestion MBP-CBD MBP CBD

16 Judith Klein-Seetharaman Thanks A CKNOWLEDGMENT S 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......