Youtor
bacterial protein overexpression
>> YOUR LINK HERE: ___ http://youtube.com/watch?v=-fzVVuGr5LE
The basic principle behind recombinant protein expression is that we can stick the genetic instructions for a protein we want made into cells from a different organism and it’ll make the protein for us. Bacteria is cheapest and easiest if it works. I can stick the genetic instructions for a protein I’m interested in into a plasmid vector (like pET28) and get the bacteria to devote almost all their time resources into making that one specific protein - I get to tell them exactly what protein I want made - and when I want it made, through IPTG induction of T7 polymerase. • blog post: http://bit.ly/bacterialproteinoverexp... • ⠀ • We can stick the genetic instructions (in the form of cDNA) for the protein we want made into a smaller piece of DNA that’s easier to work with and which has some special features. We call this carrier DNA a “vector” and for bacteria, the vector is usually a small circular piece of DNA called a plasmid. ⠀ • ⠀ • A plasmid that I commonly use for bacterial expression is pET28a(+). pET stands for p*lasmid *E*xpression vector under *T*7 control, and it’s a family of “phagemid” vectors that mix and match features of plasmids phages. T7, like other phages, has to convince the bacteria to make *their proteins instead of their own bacterial proteins. And, for recombinant expression, this is what we want to do too!. So we learn from (*steal from*) the masters. So how do they do it? It’s all about bypassing the holdups: there are 2 main “holdups” that can get in their way 1) Making mRNA copies of the gene (TRANSCRIPTION) 2) making protein from those mRNA copies (TRANSLATION)⠀ • ⠀ • TRANSCRIPTION requires an RNA POLYMERASE (RNA Pol). Bacteria have their own, but it’s busy making bacterial proteins, so rather than rely on the bacterial RNA Pol, T7 makes its own RNA Pol (T7 Pol). And this one is specific for its own PROMOTER (start site on the DNA the Pol latches onto to start making recipe copies). So T7 gets this one all to itself - the bacteria can’t use it.⠀ • We want to make LOTS of copies of the mRNA because this DOES have to compete w/bacterial mRNA for the bacteria’s protein-making machinery (RIBOSOMES). BUT because T7’s so active exclusive it can easily swamp out the bacterial mRNA.⠀ • ⠀ • If we put a T7 PROMOTER before our gene, a T7 TERMINATOR after it give it some T7 Pol, we can get bacteria to OVEREXPRESS our protein. We get the bacteria to devote almost all their resources to expressing our gene - after just a few hours over ½ of all protein in the cell could be ours ⠀ • ⠀ • BUT because the bacterial cells are devoting themselves to making our protein, they’re neglecting their own needs - including reproduction - that reason why bacteria are so useful in the lab (well, one of many reasons) is that their population booms rapidly because it doesn’t take them long to copy all their DNA (DNA replication) then split in half, giving each new cell a copy. That takes a lot of energy and resources, which the bacteria doesn’t have if it’s devoting itself to T7 protein-making. T7 doesn’t care about this, but we *do*, because we need to be able to grow the cells to get enough cells to express lots of our protein ⠀ • ⠀ • One way to do this is to just not give it T7 Pol - that special polymerase that makes the RNA copies of the T7 genes (which ribosomes use to make T7 proteins) or anything that “looks” like a T7 gene because it’s under the control of a T7 promoter (like the gene we want to express). And in fact, if you look at a pET vector, you’ll see it does NOT have the T7 Pol gene. So how does our protein get made? Don’t worry - we still have the T7 Pol gene - we just keep it separate so we can activate it “on command”⠀ • ⠀ • We rely on the bacterial host DNA and NOT the plasmid DNA to provide T7 Pol. Bacteria don’t normally have this gene (it’s from a virus that wants to sabotage it, remember), but specific strains of bacteria have been designed so they DO. If we’re still in the cloning phase only want to make more copies of the plasmid ⭕️ → ⭕️⭕️⭕️… we can stick the plasmid in bacteria that don’t have the T7 Pol gene (strains like DH5α). And then, when we want to express it, we stick it into bacteria that DO have it (like BL21(DE3))⠀ • ⠀ • BUT we still want more control - we want to be able to control when those bacteria that have the T7 Pol gene actually make T7 Pol. So we steal from another clever biological setup - the LAC OPERON, to be able to control when we express the protein More here: http://bit.ly/2MxNPs2⠀ • When we add the allolactose mimic IPTG (Isopropyl β-D-1-thiogalactopyranoside), it binds the repressor ⏩ repressor falls off ⏩ bacteria makes T7 Pol ⏩ T7 Pol binds T7 promoter in front of our gene ⏩ T7 copies the DNA into RNA until it reaches the T7 terminator they come apart ⏩ does this over over 🔁 making lots of mRNA copies that swamp out the bacterial mRNA outcompete for the limited ribosomes ⏩ ribosomes make our protein from the mRNA instructions ⏩ we celebrate! (hopefullY)⠀⠀
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