good afternoon this video is about protein purification and characterization our learning objectives for this are to be able to develop a plan to purify a protein tell why proteins are least soluble at their Pi or ISO electric Point describe protein characterization methods describe the theoretical basis of SDS page that's sodium doal sulfate polyacryamide gel electropheresis describe the theoretical Bas basis of B spectrometry for proteins describe the theoretical basis of X-ray crystallography protein purification and characterization we focus primarily on soluble proteins because they can we can purify those proteins such as collagen that's part of bone or elastin that's part of your lung or keratin part of your fingernails are very difficult to purify because they're not soluble soluble proteins are therefore easier to study uh pure protein are easier to study you can get a lot more uh really good data from Pure proteins you can't do clean work with dirty proteins so uh it's good to have a pure protein to make sure you don't have any interfering activity or interfering protein so when you measure a uh function of an enzyme for example that protein is all that enzyme and not another one that might be contaminating it proteins are drug targets so once you get a pure protein you can test it uh test drugs against it to see if they inhibit it protein purification and characterization the first thing you need to be able to do is detect and measure have a detection or measurement method for your protein uh if it's an enzyme you can use an enzyme assay in which you measure substrate disappearance or product appearance or if it's a colored protein you can measure how much of that color is there measure total protein there are various methods for doing that a real old one is called The Lowry method uh the Bradford method is based on kumas blue there's also a BCA method that's also has a different reagent in it and then there's an a280 method is just absorbance based on tryptophane tyrosine and cinee content so you choose your methods you can use solubility ion exchange Affinity Etc to possibly purify your protein we'll talk about those as we go forward and then finally once you get it you can characterize the Purity by SDS page and mass spectrometer you get your get information on the size of the protein and its sequence and then you can do amino acid sequence as well by Edmund degradation that's an old method and mass spectrometry Edmund degradation we'll talk about as well uh determine 3D structure by X-ray crystallography and now you got a beautiful picture of your enzyme or protein now proteins uh fold uh spontaneously and that folding is driven by enthropy that entropy is the release of that water these water molecules and allows them to more freely move about and the hydrophobic residues in red tend to go to the inside of a protein and the hydrophilic ones are on the outside so the water and over here you have a oil water interface that water is not free to move into this space where this hydrophobic residue is over here that water is freed up to move and that drives the process of uh INE to drive the folding reaction now enzyme aays are quite often done in 96 well microtiter plates or even larger ones and those are very useful because you can scan Wells all at once uh you can look at you can do a rate reaction on this well over time and compare it to this well they have some standards and then you can do test Inhibitors and various other Wells so it's a really unique uh easy way to do it uh most Labs have microt Micro uh tighter plate readers now uh there are even some that can read a280 if you have a plate that has that's UV visible uh it doesn't have to be quartz it can be a certain Plastics that are UV visible and you can measure protein in these as well centrifugation you can do uh differential centrifugation uh the in various ways you can isolate nuclear fractions mitochondrial fractions or microsomal fractions you can isolate GOI and various other things and that could enrich your sample for the enzyme you wish to purify and I'll show you an example of that later uh you can also dilize your protein that is when it has salt in it you can put it into a buffer with no salt salt and or water and eventually that uh whatever the ions are that are small will come out your protein which is larger be held in these bags can be a various molecular uh size exclusion usually about 12,000 um is standard dialysis tubing but you can also have various units that use semi-permeable membranes like this uh under pre nitrogen pressure Aron pressure to concentrate and dilize proteins proteins are least soluble at their is electric Point proteins uh have either a net positive charge or a net negative charge depending on the pH so at low PH they'll have a net positive charge at high pH they can usually have a net negative charge and uh in between somewhere there will be the isoelectric point or pi and that's the point at least charge solubility is lowest at this point because there's no repulsion over here positive charge the the molecules repel each other at negative charge they repel each other but at the isoelectric point there's no rep repulsion and they tend to hyro they have hydrophobic residues they'll stick together so here's a protein with its hydrophobic interactions um and those can occur because of the water molecules surrounding them however if we want to precipitate a protein quite often we add a reagent such as ammonium sulfate which competes with the water molecules the water water molecules are sequestered around the ammonium sulfate and then the hydrophobic parts of the protein could come together and it precipitates so protein aggregate via hydrophobic interactions anod exchange chromatography is based on charge so proteins can be positively charged or negatively charged and this this is also a pH dependent process as we just talked about and if you have an an anion exchanger that's a positively charged resin the negatively charged proteins and blue will bind to it and the the red positively charged proteins will be repulsed and go through and wind up in the buffer wash coming through the column after the proteins are bound you wash off anything that won't bind and then didn't you elute the protein off with Chlor with chloride you can actually use a gradient of increasing sodium chloride and separate out multiple uh NE uh positively negatively charged proteins in the process so it's a very useful method of separating proteins gel filtration separates proteins on size large ones go between the beads and little ones spend time inside the beads so the little ones come out last so the big ones come out first and then the little ones come out last in gel filtration now purification wise this is not a very useful technique uh because a lot of proteins have very similar sizes and the resolution of this is not uh great unless you have to extremely long columns but you can uh use column long columns to uh you can calibrate them with proteins of known molecular weight and then estimate the molecular weight of your protein for example a dieric protein would elop prior to its monomers Affinity chromatography is based on the biological function or activity of an protein or enzyme proteins lacking the function or Affinity do not bind examples are antibodies and antigens so here we have a column that has beads that has an antibody bound to it that recognizes uh protein recognized by the antibod is red protein not recognized by the antibody is blue so all the blue stuff flows right on through and then the red thing stick that ph7 where the antibod is functioning you can uh change conditions so that the antibody antigen complex is uh disrupted usually ph3 like tenth Moler HCL works well and then you can luch your protein over here you can also put on Inhibitors of enzymes onto these beads and they will bind certain enzymes for example you can put a Tron inhibitor on here and bind Tron and then elute that at ph3 Tron binds well at ph7 at ph3 it's binding is disrupted so you can combine these techniques into multiple ways so here is an I exchange uh profile here with a con salt concentration gradient you can see the activity of your enzyme the red line is all protein and the activity is in blue and so you pull these fractions right here and get rid of all the other stuff then you run it on gel filtration and there's some big proteins and some little proteins and you concentrate on your Peak here and then finally you do an affinity chromatography uh technique in which there are lots of proteins that are not bound and they wash through and then you change the conditions in lur and hopefully it's pure at this point so here's a uh an example example of a couple of methods for purification of the kex2 protease that's found in the ye GOI of the yeast method one there was a membrane extraction uh of uh of Lis of yeast so you lice the yeast and extracted out the membrane you had a crude membrane extract total protein 736 milligrams uh specific activity 38 that's the the uh Activity 2 280 units of activity divided by the total protein gets specific activity and we call this 100% yield for the first step and then enrichment is one here next time is deae cellulose DEA has a um positive charge on it so this is anine uh chromat tarpy anine exchange chromy and that enriches it about Eightfold and increases the specific activity to three benzamidine is an affinity method and Arginine an infin arginine sephos is an Infinity method and finally we wind up with a specific activity of about 60 wind up with 1.3 mg of protein and we've purified at 158 fold step method two uh involved the isolation of the GGI by differential centrifugation once those were isolated then they were extracted much less protein here but much higher specific activity to start with and total activity was about the same yield was quite good here relative to this technique and then deae uh surface cell is a slightly different reagent just like dea cellulose only the beads are a little different and then uh that worked out quite well high yield there uh then UTI sepharose is human urine uh Tron inhibitor and that's hook on to sephos and this is a Tron like enzyme that likes lysine and Arginine substrates and then so that one resulted in a real high Purity you got a specific activity of 260 almost four times what you had up here and a real good yield of 72% versus 27% in this this method and so the enrichment Factor was 684 here much higher than here so this method worked a lot better sometimes you have to try various methods to get there this substrate has a Paran Nitro analid so when this peptide bond between the Arginine and the Paran Nitro analy is cleaved you get this yellow product and you can measure that in a spectrometer micro P tighter plate and you're in good to go so here's another purification table and in this one you can see various methods crude extract and total protein total fite degrading activity this is units of activity specific activity purification fold and Recovery again and they did ammonium sulfate fractionation here they just uh added zero to 90% ammonium sulfate precipitated out this activity good recovery of activity here but but um not uh didn't increase the specific activity a lot just about doubled it then they did a DEA Salos then a CM cellulose this is a cat IR exchange uh technique and uh so they wound up with uh the the uh little better purif then they use uh sephus searil s200 high resolution that's gel filtration uh that worked well and then monos has a sulfate it's very NE uh very negatively charged and so that worked well as a as well as a uh as a anion exchange cat cation exchanger sorry and then you uh got a final pure protein now SDS page uh SDS will denature proteins by binding to the protein and about 1.3 grams of protein bind per gram of uh sorry 1.3 grams of SDS bind per gram of protein and SDS is as I said is sodium desal sulfate it's a 12 carbon chain with a negative sulfate at one end so that results in a uh coated protein that has a constant charge to mass ratio depending doesn't matter what the size of the protein is now slight difference some glycoproteins don't bind SDS quite the same as regular uh non-gly glycoproteins but on electris you separate by size rather than by charge because it has the constant charge Mass ratio just like DNA or RNA uh you can also use your re guanine hydrochloride to Nature proteins by hydrogen binding those are different techniques SDS uh page electropheresis uh you have a polyacryamide gel which acts as a Civ and under electropheresis the protein is positively uh is uh negatively charged so we have the negative uh cathod uh up here and we have the positive uh anode down here and since we're uh moving those anion down they migrate through here and then we can the large ones move more slowly because they SI more slowly through the gel and the and the small ones migrate faster and then you can stain them to visualize your bands you can also run colored standards so you can measure approximate sizes here relative to the ones others that are known uh here's a purification in which we have have a crude extract of some couple of purification steps and final our product here at 40,000 molecular weight nice pure looks pretty pure uh so it's a it's a way of measuring uh protein Purity as we go through a purification procedure you can also do 2D uh gel electroforesis and this is done a lot in what we call proteomics and in that process we do ISO electric focusing in One Direction Where We have a gel that with a pH gradient from four to 10 and then our protein migrates until it hits its isoelectric point and stops because it has Net Zero charge at its ISO electric point then this gel is turned and put on top of a SDS page uh gel soaked in SDS and then SDS electropheresis is run so now we SE in the second direction we separate on size charge in the first Direction and size in the second direction and you can see how many protein spots you can separate out you can actually isolate these spots and then treat them with Tron and then run them through a mass spec and determine what proteins these are kind of a neat process isn't it uh that can be that can be used to uh look at various diseases or uh different uh cell proteins and different conditions proteolysis is also an important way as I said we cleave those up with Tron so you need to know what Tron is tripson Cleaves at R lysines and arginines and uh versus choton hydrophobics and last days at aliphatic residues this is the shre Burger nomenclature for in for proteolytic enzymes and here is the polypeptide in Black the bind that gets cleaved in pink here and so this is the cleavage site so that peptide bind is going to be hydrolized this P1 residue is the most important residue usually in uh in proteins that uh this would be for Tron this would be a lysine or Arginine and then the binding pocket has an aspartic acid with a negative charge is complimentary to the B positive charges on the lysen Arginine that's why binds well in that binding pocket but notice that there are other binding pockets as well there's an S2 binding pocket for for residue P2 there's a S3 binding pocket for residue uh s P3 and there's a uh P1 Prime S1 Prime P2 Prime S2 Prime pocket and P3 Prime uh S3 Prime pocket so the prot the enzyme which is in blue makes multiple contacts with its substrate in Black uh at various primarily at these different side chain residues and therefore has spec specificity is delineated here now so you have Tron will cleave at lysine and Arginine but if it binds a lot better at P3 and P2 then it's going to cleave faster at uh if it likes these other subsite binding positions kind of an interesting concept isn't it so the enzyme doesn't make just one position this is this is the most important one but it's not the only one Edmund degradation sequencing we can sequence proteins using the Edmund chemical reaction and we use pheny isothiocyanate to do that and it reacts with the amino Terminus of a peptide and under the right conditions this process will sign to to leave this final product which is a phenol thiohydantoin amino acid and this these R groups are different and so we can identify these via hlc hlc is high performance liquid chromatography also known as high pressure liquid uh chromatography and so this process can be done and you get off the first amino acid you identify it and then you do it again with the second on the second amino acid because you freed up a free amino group here in your peptide when you cleave this off and then you can so you can do it again so you move from the inter Terminus to the c terminer one amino acid per cycle now this is a chemical reaction it only goes to about 95% completion so we sometime you can't go very far you can get maybe 10 or 20 amino acids usually uh but it's it was power it was a very powerful technique and we used it a lot back in uh many days ago and I ran one of these brute machines for several years that was was a great experience um Mass spectral analysis to identify proteins you can if you can get them to ionize and sometimes we put proteins on a matrix and hit it with a laser beam to get them to ionize then they can fly in a vacuum tube uh which is called a flight tube and they will fly based on their charge so the ones have a low higher charge Mass ratio will fly faster and they can be detected uh so the lightest ions arrived at the Protector first and the heavier ones arrive later and so you can so it's called a mass spec um don't I won't go into it any more than that uh however it's used in proteomics quite often uh if we have a cell or tissues we take out the proteins we run them on gels we isolate out a band that we thinks are is a very important en uh protein we can digest that into peptides we can run that down an hpl down an HL liquid chromator peptide separation then we can take as a coming off the column we electrospray ion ionize these things they get the ionized peptides the ionized peptides are then analyzed in a mass spec Mass spectrometer if the peptide Mass masses resulting from tip tripson digestion can be determined by Mass spectrometry using peptide Mass fingerprinting uh this will uh sometimes allow us to identify what the protein is from that because we can match this up with genomic databases and uh various other databases that expy I'll give you links to that in your handout but uh if this does not allow unequivocal identification of the protein via database searching peptides can be sequenced via tandem mass spectrometry or so show these peptides then get fragmented again in in in a mass spec uh and then those products those uh small ions get on into another Mass analyzer and then we can get the sequences so the sequence happens to be alanine glycine Lucine in this peptide so it's just as an example now Extinction coefficients of pure proteins protein to measure accurately the concentration of pure protein we'd like to know the extinction coefficient the extinction coefficient is based on the amount of tryptophane tyrosine and cinee and of course we uh this is a this uh has this form has been empirically determined and we'd like to determine the absorbance of a. 1% Solution that's 1 G per mil or 1 milligram per mil and that at 280 nanometers so BSA has an Extinction coefficient of 66 so 1 milligram per Mill solution will have 66 HSA is 0.53 and tron's 1.57 IGG imunoglobulin G is 1.43 for example here's the reference for this protein science back in 1995 and here is a link to the uh proteomics website uh the expy website and uh we're going to go there and I will uh go down to the next slide and we're going to oops I can't do sorry go back up I have to escape out of this and I'm going to copy this amino acid sequence out of here and then I'm going to go back to this and I'm going to pull this over in here and this is the proton uh Pro Pam tool at the expy website I'm going to paste that sequence right in here I'm going to hit the compute parameters and you're going to see that here's the sequence numbered sequence 260 amino acids total here's the molec here's the number of amino acids 260 molecular weight 20 29253 point9 theoretical isoelectric point is 4.58 uh this is the uh composition amount of Arginine and aspartic acid glutamic the amount of Arginine and lysine uh you can see that you have more aspartic and glutamic therefore you have the low Pi below seven and then Extinction coefficients uh here here's the molar extension coefficient so a one molar solution of this would absorb 280 nanm light at would have a 46,9 absorbance you never have a one molar solution of a protein so we deal with absorbents at 1% that's an that's 1 milligram per mil or one gram per liter and that's 1.57 very similar to Tron for example actually this is DNA uh and so we have this is useful information that you can use to understand your protein what its structure and function is and so now we're back and here's an example of a table that has uh a number of different proteins there different Extinction coefficients and here's the the a280 of a 1 millgram per Mill solution or the 0.1% solution okay so useful now x-ray crystallography uh is a once you get a pure protein you can sometimes crystallize it if you get the solubility just right and I won't go into the details of that but you can then hit that with an x-ray beam and the and the nucleus of the atoms will defract those uh x-rays those x-rays can be detected on film or now we use a uh a an instant camera like method of detecting these and you get a defraction pattern from that information on those defraction patterns uh we can uh use uh fora transform computers and very sophisticated software to create electron density maps and those electon density Maps can be converted into what looks like a uh 3D atomic model of the Protein that's quite useful to study its structure and function and here are some uh websites there are some V nice videos for this as well to explain this is the 100th anniversary of the discovery or development uh by uh doctors Bragg and Bragg Father and Son team in uh England uh they developed x-ray crystallography of very small molecules years ago and now it's been applied to proteins and here is an example of a structure spinning uh this is a model I made and this has this is human massel Chimas and it has carbohydrate chain stuck on the side we just uh this is a this is the model of a recombinant protein that we made and you can see the active cerene here 9 195 the serin at the bottom of the binding site 189 the spartic acid Nadine that are also involved in the active site and then these carbohydrates are stuck on one of them to asparagine 95 and the other one to Asar genene 72 so it's very useful method to look at um things now Dr Paul Stanton used to be president of East Tennessee State University and he was started out Life as a surgeon then a Dean then president and at graduation he always gave this quote do all the good you can by all the means you can in all the ways you can and all all the places you can at all the times you can to all the people you can as long as ever you can from John Wesley May Grace and peace be yours in abundance