1. Protein Purification Tutorial Protein purification tutorial TITLE PAGE graphic "Proteins" a word derived from the Greek word, Proteios, which means "of the first rank" was coined by Jons J. Berzelius in 1938 to stress the importance of this group of organic compounds. Proteins have been described as the servants of life. Most proteins are enzymes but in addition there are also regulatory and structural proteins.

2. Protein Purification Tutorial Intro –Why purify proteins? During the last decade, the genomic sequence of many organisms has been determined. The wealth of information in these genomes has dramatically changed research. If you can identify the gene within the genomic DNA sequence, you already know the amino acid sequence of the encoded protein and the regulatory elements that control the expression of this gene. You don’t, however, know the structural organization of the protein or how it is regulated. If you do not have a genomic database or cannot identify the protein coding region within the genome, you may want to purify the protein in order to help you to isolate the gene. –1) If you have not yet isolated the gene that encodes the protein, you may want to purify the protein for any of the following reasons: –The purified protein can be used to determine the amino acid sequence. The sequence can then be used as a probe to help in the isolation of the gene. –The purified protein can be used to produce antibodies that can be used as a probe to help in the isolation of the gene. –The purified protein can be analyzed by mass spectroscopy. The molecular weight can then be used as a screen of the genomic sequence for the gene. –2) If you have already isolated the gene that encodes the protein, you may want to purify the protein for any of the following reasons: –Pure proteins are required for structural analysis.(x-ray crystallography and NMR spectroscopy)(Figure 1). –Pure proteins are required to study enzyme function. –Pure proteins are needed in order to learn about what other proteins or nucleic acids they interacts with. (Figure 2) –Pure proteins are needed for studies of enzyme regulation (are their regulatory subunits, is it regulated by phosphorylation, is the protein regulated by interaction with other proteins.)

3. Protein Purification Tutorial Objectives In this tutorial you will: –1 –2 –3 –4 –5

4. Protein Purification Tutorial Intro – table of common techniques PropertyMethods Size / shapeSize-exclusion chromotography Isoelectricpoint (charge) Ion exhange chromatography binding to small molecules Affinity chromatography Table of common methods of protein purification solubility Precipitation with ammonium sulfate (salting out)* *Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes. It is commonly one of the first steps in a purification scheme. Purification procedures attempt to maintain the protein in native form. Although some proteins can be re- natured, most cannot! To purify a protein from a mixture, biochemists exploit the ways that individual proteins differ from one another. They differ in: –Thermal stability* *For most protein purifications, all steps are carried out at ~5°C to slow down degradative processes.

5. Protein Purification Tutorial Intro side bar Picture of protein gel with lanes showing sequential purification steps ProcedureFraction vol (ml) Total Prot (mg) Activity (units) Specific activity Units/mg Crude cellular extract 14001000010000010 Size-exclusion9040080000200 Ion exchange8010060,000600 Purification Table Add row with ammonium sulfate data. Include two colums at end called Purification factor and Yield. Purificati on factor Yield 100% 20 80% 375% 1 Note: The type and order of steps are customized for each protein to be purified. An effective purification step results in a high yield (minimal loss of enzyme activity) and large purification factor (large increase in specific activity).

6. Protein Purification Tutorial Intro – flow chart. Purification is a multi step procedure Sample Separation technique Fractionation Purification is a multi-step procedure. Is there activity? Set aside No Combine Fractions yes Monitor purity Assay total protein Assay enzyme activity Pure? Prepare for analytical technique yes No Repeat with another separation technique until pure

7. Protein Purification Tutorial Intro – first steps. First steps –1. Source. A good source is cheap and readily available. Many proteins are enriched in specific tissues, for example hemoglobin in blood. For this reason, these tissues may be excellent sources for your protein. –2. Assay: Most assays are chemical reactions catalyzed by specific enzymatic activities. Proteins that have no activity are usually assayed using SDS polyacrylamide gels.

8. Protein Purification Tutorial Assay – develop an assay First steps – Develop an Assay 1.An assay for an enzyme is a method for quantifying its activity. Since the assay is repeated many times, it is important that it be a simple procedure. Usually enzyme activity is monitored as a change in absorbance which can be measured using a spectrophotometer. For example an assay for ribonuclease measures the change in absorbance that accompanies the breakdown of RNA to ribonucleotides.

9. Protein Purification Tutorial Intro – Preparing the sample (Crude Extract) Protein from cells or tissue First steps: Preparing the sample – Crude extract. Microbial cells or tissue Break cells, tissue, or organ Blender, homogenizer, sonication, pressure, psmotic Pellet with intact cells, organelles, membranes and membrane proteins Supernatant with Soluble protein

10. Protein Purification Tutorial Segway to Chromotography Some kind of graphic. Note: In order to isolate sufficient quantities of protein, you may need to start with kilogram quantities of source tissues. These amounts can best be handled using precipitation methods (e.g. ammonium sulfate precipitation). Later in the purification, large columns can be used to handle gram to milligram quantities. Amounts handled on gels are typically in microgram quantities.

11. Protein Purification Tutorial Intro – flow chart. Purification is a multi step procedure Sample Separation technique Fractionation Purification is a multi-step procedure. Is there activity? Set aside No Combine Fractions yes Monitor purity Assay total protein Assay enzyme activity Pure? Prepare for analytical technique yes No Repeat with another separation technique until pure

12. Protein Purification Tutorial Over view of the apparatus –Glass column –Reservoir –Solid matrix – beads –Solution of protein. –Effluent. –Other terms? Column Chromotography –general case We think it would be better to have a resevoir attached to a cap on the top of the column via a plastic tube, show minimal liquid on top of the column bed, show a tube coming out of the bottom of the column that has a clamp of stopcock. Perhaps a schematic on the left and a photo of an actual column running in a cold box over a fraction collector on the right. Should emphasize that the basic set=up is the same for all column types, but the characteristics of the beads vary.

13. Protein Purification Tutorial Load sample (4 protein mix) Image of apparatus with protein mixture layered on top TEXT. The crude extract is placed on top of the solid matrix. (In this case we are using a mixture of 4 proteins, indicated by different colors.) (As the animation proceeds) The proteins move at different rates through the matrix based on the properties of the proteins and the type of column beads. Note - this should be shown as a single band (possibly brown or striped with four colors)

14. Protein Purification Tutorial Collect fractions. Fractionation Text: As the column separates the proteins in the mixture, the “effluent” drips into a series of fraction tubes that are moving at a specific rate of speed. These tubes are called fractions. Here we are showing 20 tubes. Fraction collectors in most labs have about 75-200 tubes. Be sure to remove color from column as it drips into the tubes below! If the sample is spread over three tubes, the center tube will be darker in color.

15. Protein Purification Tutorial --3 questions— In reality, proteins aren’t color- coded so we must ask ourselves these 3 questions: 1.How do we know which fractions contain protein? 2.Which of those fractions contain the desired protein? 3.How do we assess the purity? ???

16. Protein Purification Tutorial Question 1 – take A280 Screen 1. Total protein a can be estimated by taking the absorbance at 280 nm in a spectrophotometer. Aromatic amino acids absorb light at this wavelength causing all proteins to have absorbance at 280nm. Many fraction collectors measure the A280 as the column is running. Question 1. How do we know which fractions contain protein? Take A 280 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction #

17. Protein Purification Tutorial Question 1 – take A280 Screen 2. Total protein a can be estimated by taking the absorbance at 280 nm in a spectrophotometer. Aromatic amino acids absorb light at this wavelength causing all proteins to have absorbance at 280nm. Many fraction collectors measure the A280 as the column is running. The A280values can be plotted against the fraction number in is what is called an elution profile. Question 1. How do we know which fractions contain protein? A 280 Plot values 00025200025852002520 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction #

18. Protein Purification Tutorial Question 1 – take A280 Screen 3. Total protein a can be estimated by taking the absorbance at 280 nm in a spectrophotometer. The values can be plotted against the fraction number in is what is called an elution profile. Notice the peaks on the graph. These indicate where the fractions are that contain protein. Question 1. How do we know which fractions contain protein? A 280 00025200025852002520 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # A 280 Fraction # Peaks

19. Protein Purification Tutorial Question 1 – take A280 Screen 4. Total protein a can be estimated by taking the absorbance at 280 nm in a spectrophotometer. The values can be plotted against the fraction number in is what is called an elution profile. Notice the peaks on the graph. These indicate where the fractions are that contain protein. Question 1. How do we know which fractions contain protein? A 280 00025200025852002520 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # A 280 Fraction # Peaks

20. Protein Purification Tutorial Question 2 – take A280 Screen 1. Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein. Question 2. Which fractions contained the desired protein? A 280 00025200025852002520 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # A 280 Fraction # Enz. Assay. Fraction #

21. Protein Purification Tutorial Question 2 – take A280 Screen 2. Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein. Notice the results of the enzyme assay. The highest activity corresponds to one of the peaks. Question 2. Which fractions contained the desired enzyme? A 280 00025200025852002520 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # A 280 Fraction # EnzAssay Results Need to substitute values for the colored spots since we are switching to an absorbance based assay. Now we can have them discard tubes that don’t have enzyme activity.

22. Protein Purification Tutorial Question 2 – take A280 Screen 3. Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein. Notice the results of the enzyme assay. Notice that the highest activity corresponds to one of the peaks. Discard the fractions that don’t contain protein by clicking on the tubes that don’t contain protein. Question 2. Which fractions contained the desired protein? A 280 00025200025852002520 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # A 280 Fraction # EnzAssay Results

23. Protein Purification Tutorial POOL fractions – screen 1 Combine (pool) the fractions with activity. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Fraction # Fraction # Pool fractions 1.NEXT We want to pool the fractions that have enzyme activity. It may be useful to consider more than just the activity of a fraction. Specific activity is a measure of the amount of enzyme activity per amount of protein (units/mg). The higher the specific activity, the higher the purity. When pooling fractions, judgement is needed as to whether to optimize yield or specific activity.

24. Protein Purification Tutorial POOL fractions – screen 2 Combine (pool) the fractions with protein and activity. Fraction # 1.The fractions are pooled together. How do we monitor the progress of the purification?

25. Protein Purification Tutorial Assess purity – screen 1 1.Look at it on a gel. Even when overloaded, if only one band is visible, it is likely to be pure and a monomer, a homo-dimer, or a homo-multimer. If not a single band, additional bands that purify in parallel, will remain proportional throughout the purification. If a band co-purifies, it is likely to be a subunit for the enzyme. 2.Calculate the specific activity by doing a careful quantitative assay for enzyme activity/total protein. Question 3: Is this pooled sample pure? How do you monitor the progress? Standards | Crude Ext. | Pooled fractions ProcedureFraction vol (ml) Total Prot (mg) Activity (units) Specific activity Units/mg Crude cellular extract 14001000010000010 Separation method 1 9040080000200 Purification Table Results: 1.Gel shows more than one band. Since the sample is not pure, you must pass pooled sample through another separation technique Another separation

26. Protein Purification Tutorial Assess purity – screen 2 1.Look at it on a gel. A monomer should have one band. 2.Calculate the specific activity by doing a careful quantitative assay for enzyme activity/total protein. Question 3: Is this pooled sample pure? How do you monitor the progress? Standards | Crude Ext. | Pooled fractions ProcedureFraction vol (ml) Total Prot (mg) Activity (units) Specific activity Units/mg Crude cellular extract 14001000010000010 Separation method 1 9040080000200 Separation method 2 846000015000 Purification Table Results: 1.Gel shows one band 2.Specific activity is 15000. Looks good Your protein seems pure. YOU’RE DONE!!!.

27. Protein Purification Tutorial The 3 separation techniques of chromotography Intro – table of common techniques Affinity chromatography binding to small molecules Ion exhange chromatography Isoelectricpoint (charge) Size-exclusion chromotography size MethodsProperty Table of common methods of protein purification solubility Precipitation with ammonium sulfate (salting out)* Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes. It is commonly one of the first steps in a purification scheme.

28. Protein Purification Tutorial Gel filtration 1 -basis Gel filtration column chromotography separates proteins on the basis of size. We will start with 4 proteins. You will want to purify the “yellow one” 60 Kd Low pI (6) 20 Kd Low pI (7) 20 Kd Medium pI (7) 5 Kd Hi pI (8) Here’s our sample mix of proteins. Our goal is to purify protein #….

29. Protein Purification Tutorial

30. Gel filtration 2 - close up of beads The matrix of a size-exclusion chromatography column is porous beads. Run column Need two pore sizes (other size bigger than black proteins, smaller than existing pores.)

31. Protein Purification Tutorial Gel filtration 3 - run close up of column The matrix of a gel filtration column are beads with pores. The large green proteins can’t fit in pores so flows faster. The red/yellow medium sized proteins get trapped in the pores. The black small proteins stay trapped in pores longer. We’re wondering how this will work in animation. The black can permeate all pores and the space between beads, the yellow and red can permeate the space between beads and larger pores, the green will be restricted to the space between beads.

32. Protein Purification Tutorial Gel filtration 4 - zoom out Click on the peak that represents the protein of the largest molecular weight? Tubes march in from left A 280 Fraction # Be sure to keep in mind that the colors will either be in the column or in the tubes, not both!

33. Protein Purification Tutorial Gel Filtration 5. Many columns are commercially made. Here are some examples. Fig 1.1. Scanning electron micrograph of an agarose gel. Magnification x 50,000. Ref. Anders S. Medin,PhD Thesis, Uppsala University 1995. Sephadex./ This could be moved to the earlier view of the porous beads.

34. Protein Purification Tutorial ION –EXCHANGE 1 Ion-exchange column chromotography separates proteins on the basis of charge. We will start with 4 proteins. pH 7.2 pos charged column 60 Kd Low pI (6) 20 Kd Low pI (7) 20 Kd Medium pI (7) 5 Kd Hi pI (8) Need to include a slide on how to determine the charge on a protein, given its pI and the pH.

35. Protein Purification Tutorial Ion Exchange 2 – loaded proteins The matrix of an ion exchange is positively charged. What do you think will happen? pos Run column

36. Protein Purification Tutorial Ion Exchange 3 –column run The matrix of an ion exchange is positively charged. Only the pos charged proteins run through the pos charged column. The others “stick” to the column. pos These beads are porous, too, so you can show the proteins moving right through the beads in the animation, ifyou like.

37. Protein Purification Tutorial Ion Exchange 4- zoom out Only the POS charged proteins run through the column. How can we elute the other proteins? Tubes march in from left A 280 Fraction # 1 20

38. Protein Purification Tutorial Ion Exchange 5 - zoom out Increase the salt. Tubes march in from left Add salt A 280 Fraction #

39. Protein Purification Tutorial Ion Exchange 6 – after salt Increase the salt. What protein will come of the column next? Tubes march in from left - A 280 Fraction # - --- Add salt Don’t show the charges on the color spots - students should figure this out on their own!

40. Protein Purification Tutorial Ion Exchange 7 – after salt Increase the salt. What protein(s) will come of the column next? Feedback statement. Tubes march in from left --- A 280 Fraction # + --- Run column

41. Protein Purification Tutorial Ion Exchange 8 – after salt Red and yellow will have the same neg charge and will co-elute. Tubes march in from left A 280 Fraction # Salt concentration 1.5 0.0

42. Protein Purification Tutorial Ion Exchange 9 – Increase salt conc. Again. Increase the salt concentration Tubes march in from left A 280 Fraction # Salt concentration 1.5 0.0 Add salt

43. Protein Purification Tutorial Ion Exchange 10 – rum column prompt. Run the column. Tubes march in from left A 280 Fraction # Salt concentration 1.5 0.0 Run column

44. Protein Purification Tutorial Ion Exchange 11 – results. Run the column. Tubes march in from left A 280 Fraction # Salt concentration 1.5 0.0 Run column

45. Protein Purification Tutorial Ion Exchange 12 – results. Notice that 2 of the proteins eluted at the same time. Why? Is our protein pure? We were supposed to purify the red one. Tubes march in from left A 280 Fraction # Salt concentration 1.5 0.0

46. Protein Purification Tutorial Affinity Chromotography. Affinity Chromotography See notes below

47. Protein Purification Tutorial Monitoring progress. We need some info on the SDS page and the specific activity. See Jim’s hand written notes as a guide. Place final table here - we can include the thing plot with protein conc going down, enzyme amount remaining constant, and specific activity on the rise.