Why purify proteins? - UAB

1 11/10/06 Marilyn Niemann, UAB/CORD1 Why purify proteins? Detailed studies on function Determination of structure Industrial/pharmaceutical applications Generate antibodies Amino acid sequence determination1/10/06 Marilyn Niemann, UAB/CORD2 protein purification issues How much and how pure? Application Source Feasibility Native configuration? Functional/structural (yes) Microsequence (no) Antibody (maybe) Detection Method Functional assay Antigenic assay Band on gel21/10/06 Marilyn Niemann, UAB/CORD3 Affinitychromatographybinding to smallmoleculesIon exchangechromatographyIsoelectric point(charge)Size-exclusionchromatograph ySize / shapeMethodsPropertyTable of common methods of protein purificationsolubilityPrecipitation withammonium sulfate(salting out)**Ammonium sulfate precipitation is cheap, easy, and accommodates large sample is commonly one of the first steps in a purification scheme.

2 Purification proceduresattempt to maintain theprotein in native some proteinscan be re-natured, mostcannot! To purify a protein from amixture, biochemistsexploit the ways thatindividual proteins differfrom one another. Theydiffer in: Thermal stability**For most protein purifications, all stepsare carried out at ~5 C to slow downdegradative Niemann, UAB/CORD431/10/06 Marilyn Niemann, UAB/CORD51/10/06 Marilyn Niemann, UAB/CORD641/10/06 Marilyn Niemann, UAB/CORD71/10/06 Marilyn Niemann, UAB/CORD8 Define properties of target proteinand critical impurities Know your protein s structure and function pH, temperature stability (proteins mayprecipitate at pH=IEP) Effects of salt, detergent, organic solvent,metal ions Post-translational modifications that must bepresent these will affect structure andproperties51/10/06 Marilyn Niemann, UAB/CORD9 Strategy1) Select a source2) Break open cells, separate components3) Keep the protein native (usually cold)4) Develop an assay to follow the protein5)

3 Purification steps based on theproperties of the protein1/10/06 Marilyn Niemann, UAB/CORD10 First A good source is cheap and readilyavailable. Many proteins are enriched inspecific tissues, for example hemoglobin inblood. For this reason, these tissues may beexcellent sources for your Most assays are chemical reactionscatalyzed by specific enzymatic that have no activity are usuallyassayed using SDS polyacrylamide Niemann, UAB/CORD11 First steps--Develop an AssayAn assay for an enzyme is a method forquantifying its the assay is repeated many times, it isimportant that it be a simple procedure. Usuallyenzyme activity is monitored as a change inabsorbance which can be measured using aspectrophotometer. For example an assay forribonuclease measures the change inabsorbance that accompanies the breakdown ofRNA to Niemann, UAB/CORD12 Develop analytical assays for yourprotein (and impurities) protein concentration UV/Vis BCA, Bradford, Lowry ELISA protein purity/structure information SDS PAGE, HPLC, IEF, Western blot Biological activity In vitro, in vivo Impurities allergens, immunogenic proteins,endotoxins, viruses, bacteria, etc.

4 , PCR for viruses or bacteria; LAL for endotoxin;western blots or ELISAs for protein contaminants71/10/06 Marilyn Niemann, UAB/CORD13 The ELISA MethodChromogen or substrate which changes color when cleavedby the enzyme attached to the second immunoglobulin coupled to an enzyme. This isthe second antibody, and it binds to human serum which contains antibodies. If the patient isHIV+, then this serum will contain antibodies to HIV, andthose antibodies will bind to the HIV antigens on the purified, inactivated HIV antigens pre-coated onto anELISA plateNegative ELISA TestPositive ELISA Test1/10/06 Marilyn Niemann, UAB/CORD14 Western Blot Activity: BackgroundWhereas ELISA measures antibodyto whole virus and gives a"positive," "negative" orindeterminate test result, westernblotting is a more specific test. Itallows one to visualize antibodiesdirected against each viral this reason, it is a confirmatorytest for a positive HIV ELISA.

5 In anHIV Western blotting, proteins areelectrophoresed into a gel. As theproteins migrate through the gelthey are separated based uponsize and charge. Characteristically,smaller proteins migrate throughthe gel faster than larger Niemann, UAB/CORD15 Preparing the sample Crude from cells or tissueMicrobial cells or tissueBreak cells, tissue, or organBlender, hom ogenizer, sonication,pressure,psm oticSupernatant withSoluble protein Pellet with intactcells, organelles,membranes, andmembraneproteins1/10/06 Marilyn Niemann, UAB/CORD16 Before you start Define objectives for purity, activity, and quantity Define properties of target protein and criticalimpurities Develop analytical assays for your protein (andimpurities) Minimize sample handling at every stage Minimize use of additives they will have to beremoved Remove harmful contaminants early , multiplebarriers for pathogens Use a different technique at each step (multiplebarriers) Minimize number of steps (within reason)

6 91/10/06 Marilyn Niemann, UAB/CORD17 Developing a purification scheme Carry out small pilot experiments to evaluatevarious separation techniques Start with rapid high capacity techniques (whichare generally low resolution) and progress tohigh resolution low capacity techniques Minimize time and number or manipulationswhenever possible Arrange methods to minimize buffer changes ifother factors are equal Exploit unique features1/10/06 Marilyn Niemann, UAB/CORD18101/10/06 Marilyn Niemann, UAB/CORD19 Different approaches to purity Purification by removing the target molecule from thecontaminants Affinity chromatography techniques are very specific for thetarget molecule or for a group of molecules with closely relatedbiological properties. This makes them capable of fishing out the target molecule (or the group), leaving all contaminantsbehind. When applicable, these techniques are to be preferred, sincethey drastically simplify the purification protocol Purification by removing the contaminants from thetarget molecule When a suitable affinity chromatography technique is not athand, one has to rely on a sequence of general chromatographytechniques to remove the contaminants.

7 A typical purification protocol when nothing is known about thetarget protein employs the IEX-HIC-GF sequence of Niemann, UAB/CORD20 SampleSeparationtechniqueFractionationPu rification is a multi-step there ac tivity?Set asideNoCombineFrac tionsyesMonitor purityAssay total proteinAssay enzymeac tivityPure?Prepare for analytic al tec hniqueyesNoRepeat with anotherseparationtechnique until pure111/10/06 Marilyn Niemann, UAB/CORD21 Solubility of Proteins Solubility of a protein depends on ionic strength of the solution Ionic strength is defined as I = (1/2) i Ci Zi2 The sum is over all ionic species Because the charge Zi is squared, divalent and trivalent ionscontribute greatly to I. Ci is the molar concentration of species i Solubility of proteins increases as salt conc is increased, but at highsalt conc. the solubility eventually decreases. Explanation: Salt (ions) helps to diminish the charge interactionsbetween protein molecules that might cause aggregation orprecipitation.

8 At very high salt, too much water might be used to solvate the ions,leaving too little to solvate the protein , causing a loss of Niemann, UAB/CORD22 Ammonium SulfateFractionation ofProtein Mixtures121/10/06 Marilyn Niemann, UAB/CORD231/10/06 Marilyn Niemann, UAB/CORD24131/10/06 Marilyn Niemann, UAB/CORD251/10/06 Marilyn Niemann, UAB/CORD26 Note - this should be shown asa single band (possibly brownor striped with four colors)Load Sample (4 protein mix)Image of apparatuswith protein mixture crude extract isplaced on top of the solidmatrix. In this case we areusing a mixture of 4proteins, indicated bydifferent proteins move atdifferent rates through thematrix based on theproperties of the proteinsand the type of Niemann, UAB/CORD27 FractionationAs the column separates theproteins in the mixture, theeffluent drips into a series offraction tubes that are moving ata specific rate of speed.

9 Thesetubes are called we are showing 20 collectors in most labshave about 75-200 sure to remove color from column as itdrips into the tubes below! If the sample isspread over three tubes, the center tube willbe darker in Niemann, UAB/CORD28???In reality, proteins aren tcolor-coded so we mustask ourselves do we know whichfractions containprotein?2. Which of those fractionscontain the desiredprotein?3. How do we asses thepurity?151/10/06 Marilyn Niemann, UAB/CORD291 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Question 1. How do we know whichfractions contain protein ?Total protein a can beestimated by taking theabsorbance at 280 nm in amino acidsabsorb light at thiswavelength causing allproteins to haveabsorbance at fraction collectorsmeasure the A280 as thecolumn is Niemann, UAB/CORD301 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Question 1. How do we know which fractions contain protein ?

10 Total protein a can beestimated by taking theabsorbance at 280 nm in #The values can be plottedagainst the fractionnumber in is what is calledan elution the peaks on thegraph. These indicatewhere the fractions arethat contain Niemann, UAB/CORD3102520025852000252000 Fraction #A280A280 Question 2. Which fractions contained the desired protein ?EnzymeAssayResults1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Enzyme activity can bedetermined byperforming an enzymeassay on each fractionthat contains the results of theenzyme assay . Noticethat the highest activitycorresponds to one of Niemann, UAB/CORD321 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Combine (pool) the fractions with activity1. NEXT We want topool the fractions thathave enzyme activityFraction#It may be useful to consider more thanjust the activity of a fraction. Specificactivity is a measure of the amount ofenzyme activity per amount of protein (units/mg).