Baculovirus Expression Vector Systems - expressing proteins in insect cells - theory & practice скачать в хорошем качестве

Baculovirus Expression Vector Systems - expressing proteins in insect cells - theory & practice

Since all organisms, from bacteria to insects to mice to humans can read the same genetic language, you can stick the DNA instructions for making a protein into any type of cell and they can turn it into protein (we call this recombinant protein expression)- but just because they can doesn’t mean they will and just because they will doesn’t mean they’ll do it *well*! Oftentimes proteins that are more “complex” and/or require helper molecules to fold require more “complex” cells for proper expression and insect cell lines like Sf9 and Hi5 can provide that complexity needed for your protein to thrive! (at a lower cost and often higher yield than mammalian cells).⠀⠀ ⠀ blog with full text (text old, also has static graphics): https://bit.ly/baculovirusexpression Baculoviral vectors allow me to turn an insect virus into a vehicle for delivering the genetic recipe for a protein I want to get lots of to purify and study. quick terminology note on what we call those genetic recipes: A gene is a stretch of DNA in a chromosome which contains instructions for making some sort of product, such as a protein or a functional RNA. To actually make a protein based off of those genetic instructions, our cell first makes RNA copies of them, and then removes regulatory regions in a process called splicing. With some further processing (e.g. capping & tailing) you get mature messenger RNA (mRNA) which is what the protein-making machinery (ribosomes) use as instructions for making proteins. For recombinant protein expression, we want to give cells these edited versions, but we need them to be in DNA form so that we can recombine them with our DNA vector. So we use cDNA(short for complementary DNA) which is a DNA version of the mature, edited, mRNA copy of the gene. But we often just (sloppily) talk about putting “genes” in and assume that we’re really talking about putting cDNA in. It’s one of those things that you really want to make sure you do right when designing the clone, but in general talk, it’s way easier to just say “gene” and tends to be more follow-along-able so I do it a lot. ⠀⠀ ⠀⠀ Bacteria can read & write in the universal genetic language, so they can take those genes and make protein following their instructions (each 3-letter RNA “word” specifies a specific amino acid (protein letter) to be added). However, bacteria they have different machinery so sometimes they can’t properly fold and/or or modify proteins that are normally expressed in different types of cells. Bacteria are what we call PROKARYOTIC - they don’t have conventional membrane-bound compartments inside their cells. We, and other plants and animals are EUKARYOTIC - we do have such cordoned-off rooms in our cells, including a membrane-bound nucleus, where we keep our DNA. And that’s just one of the many ways we differ. ⠀⠀ ⠀⠀ Insect cells are closer to our cells - they have more similar machinery - but they’re easier (and cheaper) to grow than mammalian cells, so they’re a good “next try” if bacterial expression doesn’t work out. We usually try expressing a protein in bacteria first because it’s way easier, quicker, & cheaper. But that’s only if it works - if doesn’t work well you waste a lot of time, effort, and cash!⠀⠀ ⠀⠀ Bacterial recombinant protein expression is a lot easier because you just stick the gene (technically cDNA, remember) for the protein you want into a circular piece of DNA called a plasmid vector → stick that vector into bacteria → tell them to make it and they will. More on that here: http://bit.ly/bacoverexpression ⠀⠀ ⠀⠀ For insect cell expression, you still stick the gene into a plasmid, but then you combine that plasmid with another one that has insect-virus-making instructions to make a BACMID → then you isolate that bacmid and stick it in insect cells → those insect cells start making & secreting BACULOVIRUS → then you isolate that virus (V0) and add it to more insect cells → those cells start making & secreting MORE BACULOVIRUS → then you isolate that virus (V1) & add it to TONS of insect cells for LARGE—SCALE EXPRESSION → those cells start making PROTEIN! → isolate & purify the protein. Don’t worry, more detail than you probably want below! ⠀⠀ “Large-scale” can mean different things, depending on how well the protein you’re interested in expresses, how much of the expressed stuff you can expect to lose during purification, and how much pure protein you need. For most of my preps, I express about 4 L and end up with a few mgs at the end of the multi-step purification process. For industrial purposes, they can do it in giant tank things called bioreactors because they need A LOT! But, except in a few cases when I’ve used a “WAVE bag,” which can hold ~8L, I usually express in flasks which hold up to a liter at a time in. http://bit.ly/2OhWL9t⠀⠀ see blog for full version: https://bit.ly/baculovirusexpression