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Method Article
Expression and purification of recombinant human L-OPA1 using BmNPV bacmid-silkworm expression system
Naotada Ishihara
Ishihara lab, Kurume University
Corresponding Author
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This work is licensed under a CC BY 4.0 License. Read Full License
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.
Keywords
mitochondria, membrane fusion, dynamin-like GTPase, BmNPV bacmid-silkworm expression system
Xiang Zhang
commented on 22 May, 2019
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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Associated Publications
Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin
by Tadato Ban, Takaya Ishihara, Hiroto Kohno, +6
Nature Cell Biology (20 June, 2017)
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This is a preprint. It has not completed peer review.
Method Article
Expression and purification of recombinant human L-OPA1 using BmNPV bacmid-silkworm expression system
Naotada Ishihara
Ishihara lab, Kurume University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 20 Jun, 2017
Community comments: 1
Metrics
Comments: 1
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
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.
Xiang Zhang
commented on 22 May, 2019
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'
Associated Publications
Molecular basis of selective mitochondrial fusion by heterotypic action between OPA1 and cardiolipin
by Tadato Ban, Takaya Ishihara, Hiroto Kohno, +6
Nature Cell Biology (20 June, 2017)