Re: [DIYbio] More beginner questions

That's essentially his-tagging with a nickel column, which is the most widely used purification method. Good foresight.

If you're just looking to purify something that is, say, thermophilic, it's much easier. Just boil all the proteins and purify intact protein. Also, usually you add IPTG to get proper overexpression. In normal expression strains, addition of IPTG activates T7RNAp gene, which then transcribes your gene. Also need general selection marker on the plasmid. Avoid amp. Even though it is most common, it will sometimes lower your total purified protein because cells can avoid selection.

-Koeng

On Wednesday, May 4, 2016 at 7:20:49 PM UTC, Strat-o wrote:

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!

--
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diy...@googlegroups.com. To unsubscribe from this group, send email to diybio+un...@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To unsubscribe from this group and stop receiving emails from it, send an email to diybio+un...@googlegroups.com.
To post to this group, send email to diy...@googlegroups.com.
Visit this group at https://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/a390aa0d-6be1-4671-8747-cce23875b8ba%40googlegroups.com.
For more options, visit https://groups.google.com/d/optout.

--

----------------------------------------
Brian Degger

twitter: @drbrian

http://makerspace.org.uk

http://transitlab.org

----------------------------------------

--
-- You received this message because you are subscribed to the Google Groups DIYbio group. To post to this group, send email to diybio@googlegroups.com. To unsubscribe from this group, send email to diybio+unsubscribe@googlegroups.com. For more options, visit this group at https://groups.google.com/d/forum/diybio?hl=en
Learn more at www.diybio.org
---
You received this message because you are subscribed to the Google Groups "DIYbio" group.
To unsubscribe from this group and stop receiving emails from it, send an email to diybio+unsubscribe@googlegroups.com.
To post to this group, send email to diybio@googlegroups.com.
Visit this group at https://groups.google.com/group/diybio.
To view this discussion on the web visit https://groups.google.com/d/msgid/diybio/93befa5f-edb8-4cb5-b524-f8280c3dad8f%40googlegroups.com.
For more options, visit https://groups.google.com/d/optout.