Скачать с ютуб Fusion partners for improving solubility & expression of recombinant proteins - SUMO, GST, MBP, etc. в хорошем качестве

Fusion partners for improving solubility & expression of recombinant proteins - SUMO, GST, MBP, etc.

Fusion partners like SUMO, GST, or MBP, are little proteins that cells love to make that you tag on to the end of the protein you’re trying to get the cells to make, a bit like a “foot in the door” marketing campaign for recombinant protein expression! Fusion partners can help your protein of interest express itself, protect it from harm, & stick with it as they try to find their “best self” (fold properly). They’re one, but not the only, way to try to improve the expression of soluble protein. I have talked a lot about other methods before, but found for fusion partners, I want to tell you more! blog form: https://bit.ly/fusion_partners note: adapted from longer past post which has more on other strategies to try too: http://bit.ly/expressionoptimizing So today I thought I’d go over some background and practical advice for things you can do if you ask a cell to make a protein for you and it refuses (or your protein ends up misfolded and clumped up in insoluble inclusion bodies). The post has an emphasis on fusion partners, but I also go into other tips and tricks to try.    Proteins are chains of amino acids that fold up into functional (and beautiful) 3D shapes. The sequence of amino acids in the chain is determined by the gene. The gene (DNA form) is copied into an RNA version (mRNA) (transcription) then that mRNA is used as instructions to link together the right amino acids. When we express proteins RECOMBINANTLY, we stick the DNA instructions for a protein into an easy-to-work-w/circular piece of DNA called a PLASMID VECTOR (this is the recombining part) then stick that into cells (often harmless bacteria or insect cells) to make the protein for us (this is the expression part). http://bit.ly/proteincleaning    We’re in control of the DNA we put in there, which means we’re in control of the sequence of amino acid protein letters in the protein that gets made (we can even add on extras like affinity tags and fusion partners). But the expression cells are in control of whether that protein actually gets made - and made correctly (though we have ways to try to coax them to).    When you go to purify the protein (assuming you’re purifying a non-secreted, non-membrane protein (i.e. a soluble cytoplasmic protein)), you break open the cells (lyse them), spin that lysate super fast in a centrifuge to pellet out the insoluble stuff (membrane bits, etc.), and then purify the protein of interest from the liquid part (supernatant) containing the soluble things. If you’re trying to purify a protein and you “can’t find it” there are a few possible culprits  - the protein wasn’t expressed (the mRNA didn’t get translated)  - the protein expressed but not “properly” so it’s hidden with the insoluble membrane gunk you pelleted out  - the protein was expressed but then degraded  - your ID method is flawed (problem with the purification if that’s how you’re checking or antibodies if you’re checking via western blot)    Later in the post I will get into how to check for these various things (and hopefully prevent or correct the problems). But for now let’s talk mostly about the solubility issue.   During translation, the amino acids are linked (w/help of ribosomes) 1 at a time to the end of a growing chain, going from N terminus to C-terminus. The protein chain starts folding as it emerges from the ribosome’s “chimney,” sometimes with the help of proteins called chaperones. Correct folding of the protein is really important for SOLUBILITY! SOLUBILITY is whether each molecule of something is fully coated in water. Properly folded proteins should be soluble (or embedded in a lipid membrane if they’re a membrane protein). Proteins are able to be soluble because they fold so that parts of the protein that like to interact with water (are HYDROPHILIC) are on the outside & parts that don’t like water (are HYDROPHOBIC) are sequestered away in the center. more on solubility (something we crystallographers think about a lot): http://bit.ly/solutionconcentrations   If a protein doesn’t fold properly, those water-hating hydrophobic parts get exposed to water & panic - so they stick to the hydrophobic parts of other misfolded proteins (because that’s better than being next to water) & thus they clump up & aggregate. This is a helpful way to think about it, although technically, it’s more like the water molecules don’t like them so the water molecules do whatever they can to link to each other instead or your protein, which forces the hydrophobic protein parts together - this the hydrophobic exclusion effect and you can learn more about it here: http://bit.ly/hydrophobesarenotafraid   finished in comments