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Team:Cambridge/Project/In Vitro
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Purification of recombinant reflectin from bacteria was necessary in order to perform all of the subsequent ''in vitro'' studies of the protein. An over-exressing vector construct was used to maximise yield, and a his-tag was used for purification purposes. | Purification of recombinant reflectin from bacteria was necessary in order to perform all of the subsequent ''in vitro'' studies of the protein. An over-exressing vector construct was used to maximise yield, and a his-tag was used for purification purposes. | ||
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==Tagging the reflectin gene== | ==Tagging the reflectin gene== | ||
Revision as of 09:26, 13 September 2011
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Contents |
Objective Two - Isolate and Purify Reflectin in vitro
Purification of recombinant reflectin from bacteria was necessary in order to perform all of the subsequent in vitro studies of the protein. An over-exressing vector construct was used to maximise yield, and a his-tag was used for purification purposes.
Tagging the reflectin gene
The reflectin genes were his-tagged by incorporating the his sequence into the primers used to clone the gene. His tagging was chosen because...
Overexpression
We chose to use a high copy plasmid with a pBAD promotor in order to overexpress reflectin. This produces inclusion bodies, which will create unfolded reflectin, but this is not a concern because the solvents
