Expression and purification of recombinant human L-OPA1 using BmNPV bacmid-silkworm expression system
This protocol describes a methods for expression and purification of human recombinant L-OPA1 using the BmNPV bacmid-silkworm expression system. Using this method, we have shown how the dynamin-like GTPase and membrane lipids mediate membrane fusion in mammals in associated publication ”Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin” in Nat. Cell Biol..
Because bacmid preparation and silkworm culture is easy, and the bacmid-silkworm expression system might have advantages for the expression of large membrane proteins in comparison with Escherichia coli expression system. the present method will have general applicability to various mitochondrial membrane proteins. It takes 5 days for bacmid preparation, 7 days for protein expression, 2 days for protein purification and 1 days for detergent exchange, respectively.
M13 forward (-40) 5′-GTTTTCCCAGTCACGAC-3′ M13 Reverse 5′-CAGGAAACAGCTATGAC-3′ I used this pair of primers and there was primers dimer. And I think M13 forward TTTCC and reverse GGAAA would easily form dimer. Why not use the recommended primers that Gibco provide in its Users guide which are pUC/M13 Forward 5'-CCCAGTCACGACGTTGTAAAACG-3' pUC/M13 Reverse 5'-AGCGGATAACAATTTCACACAGG-3'
Posted 20 Jun, 2017
Expression and purification of recombinant human L-OPA1 using BmNPV bacmid-silkworm expression system
Posted 20 Jun, 2017
This protocol describes a methods for expression and purification of human recombinant L-OPA1 using the BmNPV bacmid-silkworm expression system. Using this method, we have shown how the dynamin-like GTPase and membrane lipids mediate membrane fusion in mammals in associated publication ”Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin” in Nat. Cell Biol..
Because bacmid preparation and silkworm culture is easy, and the bacmid-silkworm expression system might have advantages for the expression of large membrane proteins in comparison with Escherichia coli expression system. the present method will have general applicability to various mitochondrial membrane proteins. It takes 5 days for bacmid preparation, 7 days for protein expression, 2 days for protein purification and 1 days for detergent exchange, respectively.
M13 forward (-40) 5′-GTTTTCCCAGTCACGAC-3′ M13 Reverse 5′-CAGGAAACAGCTATGAC-3′ I used this pair of primers and there was primers dimer. And I think M13 forward TTTCC and reverse GGAAA would easily form dimer. Why not use the recommended primers that Gibco provide in its Users guide which are pUC/M13 Forward 5'-CCCAGTCACGACGTTGTAAAACG-3' pUC/M13 Reverse 5'-AGCGGATAACAATTTCACACAGG-3'
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