Re: [DIYbio] More beginner questions

4:09 AM |

In short its complicated, but it is a good question. 

1. depends on the plasmid construct, but generally yes. One  typical example. is to have a IPTG inducible promotor [search:  iptg inducible promoter] next to the dna for the protein of interest. In this case you grow the e. coli until you get to a suitable high density, then you switch on expression with the IPTG.
2. depends on the plasmid again....some may pump the protein into the media, some might deposit the protein in inclusion bodies that need to be freed from the rest of the bacteria by smashing them. Depending on the protein it may not be active. in general it needs a lot to purify it. 

Here is a paper from a quick search that describes one strategy. 

Soluble Expression of Disulfide Bond Containing Proteins FGF15 and FGF19 in the Cytoplasm of Escherichia coli
Bo Kong, Grace L. Guo 

Cheers,
Brian
      

On Tue, May 3, 2016 at 5:01 AM, Strat-o <marlin.mixon@gmail.com> wrote:
Starting to develop a keen interest in this topic.  First post.  

Let's say I have a viable bacterium (with my protein of interest inserted into a plasmid) and i inoculate a suitable growth media with my e. coli.  

1. Do I need to anything special to ensure that my protein of interest gets expressed?  
2. After the growth phase has run its course, how to i extract my protein of interest?  (I assume there's not just one way, just interested in approaches to purification)

Thanks!

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Brian Degger
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