Re: [DIYbio] More beginner questions

Thanks for that, Brian.  So purification sounds like it is a difficult problem.  Here's my thoughts on solving it:  Add a monomer (disclaimer, I suck at chemistry and associated terminology but please bear with me).  The monomer has a "key" that will somehow lock in the target protein.  You stir it up and all the monomers now are attached to all the proteins.  Then you add a catalyst or heat or something and the monomers all become a polymer glob.  Then you pull out the glob and wash away everything else from it.  Then you have another catalyst or maybe a higher temperature that causes the polymer to release the proteins.  How hard would it be to get something like that to work?

On Tuesday, May 3, 2016 at 6:10:16 AM UTC-5, DrBrian wrote:

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 

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0085890

Cheers,

Brian

      

On Tue, May 3, 2016 at 5:01 AM, Strat-o <marlin...@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

twitter: @drbrian

http://makerspace.org.uk

http://transitlab.org

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