Chapter 10 Protein Isolation and Purification

Transcription

1 Chapter 10 Protein Isolation and Purification 1 Introduction 2. Assay for activity 3. Homogenization and fractionation 3.1 Mechanical Disruption 3.2 Liquid Homogenization 3.3 Sonication 3.4 Freeze/Thaw 3.5 Mortar and Pestle 3.6 Additives/Facilitators 3.7 Disadvantages of Traditional Lysis Methods 3.8 Fractionation 3.9 Centrifugation 3.10 Dialysis 4. Protein Quantitation 4.1 Lowry assay 4.2 Bradford Assay: 5. Chromatography 5.1 Gel filtration Chromatography 5.2 Ion exchange chromatography 5.3 Affinity chromatography 5.4 HPLC / FPLC 6. Electrophoresis 6.1 SDS-PAGE Gel 6.2 Isoelectric focusing 7 Sequencing 7.1 Automatic Sequencer 7.2 MALDI-TOF-MS

2 1 Introduction Protein isolation and purification from cells is vital to the study of protein characterization. The synthesis of any polypeptide over 15 residues long is unlikely to give good yield. the simple math even when you have 95% binding in a bench top synthesis of proteins is (0.95)^15 = 46% yield expand that even a small protein of 100 residues long will give you a 0.59% yield in an ideal setup. But conveniently for us nature has a pretty good system for making proteins. Proper tools and modern biotechnology techniques make getting proteins as easy as going to Ikea. But the key to this is getting a good yield of protein during the isolation and purification steps. In this chapter we discuss some steps and techniques to do just that. FPLC setup

3 2. Assay for activity Activity is important for our protein. That is precisely how we will know not only the mass of our yield but its health. Some of the particulars of the isolation can lead to degradation of the protein of interest. Monitoring the activity during the various steps of the isolation can be beneficial in order to not waste time and resources. As with many of the particulars in these chapter the most important thing to know is that your protein of interest will be the deciding factor in what techniques that you need to use.this step in particular is all based around what the protein was designed to do. If the protein job is to preform a hydrolyze a bond when cofactors are present then that is the assay for the activity. D-amino acid Oxidase for example is found in many organisms. D-Amino-acid oxidase catalyzes oxidative deamination of D-amino acids (stereoisomers of naturally occurring L-amino acids) to the corresponding 2-oxo acids, producing ammonia and hydrogen peroxide in the course of the reaction (Ryuichi Konno 1998). The assay to dermic activity ti s to catalyze the reaction of d-amino acid oxidase. in this particular case the deaminated product can then be reacted to yield a colorimetric result to identify the activity. in the figure given we show the assay for the lactate dehydrogenase. 3. Homogenization and fractionation The basic goal of this part of the process is to free the protons from the cells that they are contained by braking the cell membranes and walls. This will be the goal of many of the techniques that that will be described here. First and foremost the particulars f the protein of interest need be known. for example if the protein is particularly sensitive to temperature fluctuations the analyst needs to keep the in mind. If the protein i heat stable that will give an advantage to the purification technique. Thermo scientific gives a good description of the different methods they support. adapted and modified form that is the descriptions below.

4 3.1 Mechanical Disruption Mechanical methods rely on the use of rotating blades to grind and disperse large amounts of complex tissue, such as liver or muscle. This is much like a household blender but different adaptations have been made in particular the surface area that has exposure to the cells. 3.2 Liquid Homogenization A French press consists of a piston that is used to apply high pressure to a sample volume of 40 to 250 ml, forcing it through a tiny hole in the press. Only two passes are required for efficient lysis due to the high pressures used with this process. The equipment is expensive, but the French press is often the method of choice for breaking bacterial cells mechanically.

5 Liquid-based homogenization is the most widely used cell disruption technique for small volumes and cultured cells. Cells are lysed by forcing the cell or tissue suspension through a narrow space, thereby shearing the cell membranes. Three different types of homogenizers are in common use. A Dounce homogenizer consists of a round glass pestle that is manually driven into a glass tube. A Potter-Elvehjem homogenizer consists of a manually or mechanically driven PTFE (teflon) pestle shaped to fit a rounded or conical vessel. The number of strokes and the speed at which the strokes are administered influences the effectiveness of Dounce and Potter-Elvehjem homogenization methods. Both homogenizers can be obtained in a variety of sizes to accommodate a range of volumes. 3.3 Sonication Sonication is the third class of physical disruption commonly used to break open cells. The method uses pulsed, high frequency sound waves to agitate and lyse cells, bacteria, spores and finely diced tissue. The sound waves are delivered using an apparatus with a vibrating probe that is immersed in the liquid cell suspension. Mechanical energy from the probe initiates the formation of microscopic vapor bubbles that form momentarily and implode, causing shock waves to radiate through a sample. To prevent excessive heating, ultrasonic treatment is applied in multiple short bursts to a sample immersed in an ice bath. Sonication is best suited for volumes <100 ml. Shown is my sonication probe. This is a bench-top method.

6 3.4 Freeze/Thaw The freeze/thaw method is commonly used to lyse bacterial and mammalian cells. The technique involves freezing a cell suspension in a dry ice/ethanol bath or freezer and then thawing the material at room temperature or 37 C. This method of lysis causes cells to swell and ultimately break as ice crystals form during the freezing process and then contract during thawing. Multiple cycles are necessary for efficient lysis, and the process can be quite lengthy. However, freeze/thaw has been shown to effectively release recombinant proteins located in the cytoplasm of bacteria and is recommended for the lysis of mammalian cells in some protocols. 3.5 Mortar and Pestle Manual grinding is the most common method used to disrupt plant cells. Tissue is frozen in liquid nitrogen and then crushed using a mortar and pestle. Because of the tensile strength of the cellulose and other polysaccharides comprising the cell wall, this method is the fastest and most efficient way to access plant proteins and DNA Additives/Facilitators Cells can be treated with various agents to aid the disruption process. Lysis can be promoted by suspending cells in a hypotonic buffer, which cause them to swell and burst more readily under physical shearing. Lysozyme (200 µg/ml) can be used to digest the polysaccharide component of yeast and bacterial cell walls. Alternatively, processing can be expedited by treating cells with glass beads in order to facilitate the crushing of cell walls. This treatment is commonly used with yeast cells. Viscosity of a sample typically increases during lysis due to the release of nucleic acid material. DNase can be added to samples (25-50 µg/ml) along with RNase (50 µg/ml) to

7 reduce this problem. Nuclease treatment is not required for sonicated material since sonication shears chromosomes. Finally, proteolysis can be a problem whenever cells are manipulated; therefore, protease inhibitors should be added to all samples undergoing lysis. 3.7 Disadvantages of Traditional Lysis Methods Although physical methods have traditionally been used to disrupt cells, there are some inherent disadvantages to their use. Localized heating within a sample can occur with many of the techniques described, leading to protein denaturation and aggregation. To avoid this problem it is essential to pre-chill equipment and keep samples on ice at all times. Reproducibility with homogenization and grinding methods can be challenging due to inexact terminology used to define sample handling. Furthermore, cells disrupt at different times so the viscosity of the medium constantly changes, and released sub-cellular components are subjected to disruptive forces. In addition to sample handling problems, some physical disruption methods require fairly expensive equipment, such as the French press and sonicator. 3.8 Fractionation Fractionation can be done by a number of methods depending on what the protein requires. Heat stable protease can sustain temperatures higher then normal potions can stay intact. One method to fractionate is to heat the cell lysis up to close to boiling temperatures for about 20 min. This would cause many of the protein to aggregate and will be able to be separated gravometrically. Salting out is another useful method for fractionating the cells by increasing the Ammonium Bicarbonate concentration of the solute different proteins will crash out of solution because of the charge of the protein and the concentration of the solution the proteins have differing solubility.

8 3.9 Centrifugation Centrifugation is an important part of the process of purification. Centrifugation can separate gravometrically different parts of the samples. this is useful when in conjunction to the cell lysis or the fractionation. this will give differential separation based on the mass or density. Centrifugation can be used with a sucrose gradient to separate membrane bound proteins. The gradient will yield different environments of density for the membranes of interest lingo up with. ii done right this can eliminate many steps that would make it difficult to separate other proteins bound to membranes that may have similar chemical character.

9 Ultra-centrifugation is a technique where extremely high gravity is exposed to a sample to make even more precise separations based on the differential density of the species

10 3.10 Dialysis Dialysis is sometimes necessary to remove salts that will interfere with further protein purification techniques or activity. basically dialysis involves the salt concentration decreasing through diffusion though a semi-permeable membrane. Dialysis generally takes time and repetitions. This may take 4-6 hours for each equilibration and 2-4 equilibrations may be necessary. 500mM Salt with a ratio of 50 ml to 2000 ml would yield a reduction to 2.5% of the original salt concentration. so 500*(0.025)^3 = 0.78 mm salt.

11 4. Protein Quantitation Protein quantitation i used to show the amount of protein the we have in our sample. this is important in order to plan experiments and to know what mass is just extra that we have carried along during our purification one of the major assays to determine protein content is the Lowrey assay and the other main assay is the Bradford Assay. 4.1 Lowry assay Lowry assay as described in class notes: The sensitivity of the procedure of Lowry is moderately constant from protein to protein, and it has been so widely used that Lowry protein estimations are a completely acceptable alternative to a rigorous absolute determination in almost all circumstances where protein mixtures or crude extracts are involved. The method is based on both the Biuret reaction, where the peptide bonds of proteins react with copper underalkaline conditions producing Cu+, which reacts with the Folin reagent, and the Folin- Ciocalteau reaction, which is poorly understood but in essence phosphomolybdotungstate is reduced to heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic amino acids. The reactions result in a strong blue color, which depends partly on the tyrosine and tryptophan content. The method is sensitive down to about 0.01 mg of protein/ml, and is best used on solutions with concentrations in the range mg/ml of protein.

12 4.2 Bradford Assay: Bradford, M. Anal. Biochem. (1976) 72, This assay is also known as the coomassie blue stain. This stain interacts with the peptide bond in proteins and chains from a red to a blue that is sensitive to the microgram range. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range. 5. Chromatography 5.1 Gel filtration Chromatography Gel filtration chromatography is also known is size exclusion chromatography. In this method the analyze is going though the column and being separated based in the size. the column packing had different sized beads with various pore sizes. the theory behind this separation is that the path length the smaller molecules will travel though will be greater the the path length of the larger molecules. the sailor molecules will travel in and out of the pores and crevices tint the silica beads where the larger ones may not enter all of the pores and the largest ones will not have much retention on the column at all. This method is best when used to separate molecules with molecular masses of 2 or 3 times greater size such as aggregates.

13 5.2 Ion exchange chromatography In ion exchange chromatography the separation occurs not merely by the flow of the mobile phase but also the chemical character of the mobile phase as it relates to the eluent. The chemistry behind this separation is the molecule of interest if first loaded onto the column. Then as it travels to the column it is retained by try charge of the molecule. If our protein has a majority of negative charges the stationary phase will be positively charged. When the positive charge stationary phase is exposed to the protein they associate and stick to the column packing. As a gradient of increasingly negatively charged mobile phase is washed through the column the column packing finds an affinity to the mobile phase over the protein and then the protein is eluted through the column. This is often useful when dealing with proteins with a significant charge. 5.3 Affinity chromatography affinity chromatography is used when the protein has a chemical (biochemical) characteristic that can be exploited. one of the most common is the his-tagged protein purification. when the protein is being expressed the His-tag is added (5 histidines is added to the sequence of the polypeptide). This polypeptide has an affinity to a nickel 2+ column. This will allow only the protein of interest to be bound to the column and then eluted by itself for easy purification. This is one of the preferred methods for quick isolation or proteins.

14 5.4 HPLC / FPLC HPLC stands for High performance liquid chromatography. Most of the techniques discussed can be applied to the HPLC which uses a pump to make the system run more efficiently at high pressure rather than just gravity. One of the most common form of this is known as liquid / liquid chromatography. The stationary phases has a liquid character. This is accomplished by having a hydrophobic chain bound to a silica bead. This allows the molecular scale fluid nature to interact with the proteins and peptides but the stationary characteristics that make the separation possible. This type of separation is known as reverse phase liquid chromatography because the hydrophobic character of the protein makes it more soluble in the carbon chain coated silica then the hydrophilic mobile phase A. As a gradient of hydrophobic mobile phase is added the proteins have an increasing solubility in the hydrophobic mobile phase and will separate. Where HPLC is run under higher pressures and gives very precise separations FPLC is known as Fast Performance Liquid Chromatography. This technique is just like HPLC inly that instead it uses lower pressure and different types of columns. This technique id focused on more large scale separations where precision is sacrificed for the ability to separate more protein quickly.

15 6. Electrophoresis 6.1 SDS-PAGE Gel The SDS PAGE gel stands for Sodium dodecyl sulfate- Poly acrylimide gel electrophoresis. The technique separates much like the size exclusion chromatography only on a gel and with slightly different twist. It is the same in the fact that pore size of the gel is the key to separation burt instead of the flow of a mobile phase there is an electrical current. The one interesting thing about this technique is that the proteins will all travel at the same rate/molecular mass. The SDS makes this possible. It will form a micelle around the protein giving all proteins a consistent radius per unit mass. This makes all fold of protein on a common playing ground. This is a very rapid separation technique that can yield constant and sensitive results and help track the purification process.

16 6.2 Isoelectric focusing Isoelelectic focusing as adapted form the class notes relies on the behaviour of a group of small buffer molecules termed ampholytes. These ampholytes migrate rapidly in the presence of the electric field and distribute themselves at a certain point in the electric field. These ampholytes create a local ph gradient (because of their weak acid or base nature), and by mixing several different ampholytes with different phs, a gradient of ph can be created in the gel or column. Protein placed in the system will now migrate according to its net charge, but it will stop migrating when it reaches a ph that corresponds to its isolelectric point (pi). If the protein is unmodified (by e.g. phosphorylation), the pi will be very sharply defined and hence the "focusing" in the name. For IEF, bands must be cut out of the gel at the end of the run; for chromatofocusing, they can be eluted after or during the focusing. Proteins can then be separated based on their pi values. ph gradient: polyampholytes with many pi values differ by 0.01.

17 7 Sequencing 7.1 Automated sequencing Sequenced through the edam degradation method will tell you their particular sequence of the protein that you have. this helps with the characterization. especially if you are characterizing a protein the was just discovered and little i known about. the sequences is the first piece of information the you can find and start understanding the nature of the molecule. Most of the sequencing is automated with instruments that make the process smooth and efficient.

18 7.2 MALDI-TOF-MS Matrix assisted laser desorption ionization - time of flight - mass spec The mass spec is used to determine the mass of the protein of interest to verify the identity of the fractions that are separated. Protein isolation and purification is a topic could be expanded upon but this should server as a primer to get you started. Please me at jharrold@rutgers.edu if you would like any more information. Good Luck!! --John Harrold