Overview of Protein PurificationThis laboratory manual outlines the procedures for the purification of an enzyme, ß-galactosidase, from a strain of E. coli cells. Extracting and purifying this protein from E. coli will be a multi-step process, starting from cell paste of E. Coli, progressing through several separation methods, and ending with analysis and verification of the purified protein by polyacrylamide gel electrophoresis (PAGE) and Immunoblotting procedures. Unlike many classroom laboratory activities, purification of ß-galactosidase is a project which will span the entire semester. The project is designed, as much as possible, to simulate a “real world” biotechnology experience in order to give you the skills you need to do similar projects on the job. Laboratory notebook documentation is always important, but becomes even more important in a project environment. Written and oral communication skills will be emphasized in several ways – by laboratory notebook documentation, individual progress reports and by an oral presentation summarizing the project. Teamwork skills will be emphasized by working with the same lab partner all semester and by collaboration with other students to give the oral presentation. What is protein purification? Protein purification is the separation of a specific protein from contaminants in a manner that produces a useful end product. What is a protein? Proteins are the major components of living organisms and perform a wide variety of essential functions in cells. Many of the products that are produced in modern biotechnology companies are proteins. These proteins may be drugs, such as insulin or tissue plasminogen activator (TPA), or they may be the molecular tools that enable biotechnology research such as the enzymes that are used to cut and paste DNA. In biochemical terms, each protein is composed of varying numbers and kinds of 20 different amino acid building blocks. The sequence of amino acids determines the relative positioning of individual chemical groups of each amino acid which in turn determines the final structure and function of the molecule. Protein structure and function will be explored in more depth in Chapter 2. Why purify proteins? Technicians and scientists in biotechnology must face the challenge of protein purification regularly. In a research environment, proteins must be purified in order to determine their structure and study their biochemical properties. In industrial settings, proteins are purified on a larger scale in order to be sold as products such as drugs, vaccines, diagnostic tools or food additives. Protein purification is a challenge. Protein purification is a challenge because, in addition to the target protein that you want to purify, the cell contains several thousand other proteins along with nucleic acids (DNA and RNA), polysaccharides, lipids, and small molecules. A target protein for purification may constitute as little as 0.001% of the total protein in the cell. At most, it may be 10%, unless over-expression is induced by genetic engineering methods- in that case, it may be expressed at higher levels, up to 60% of the total cell protein. In any case, the challenge is to separate the target protein from all the other components in the cell with reasonable efficiency, yield and purity. The two most important things required to make a purification possible are a specific assay for the target protein and a means of stabilizing it during the purification. Separation methods are based on protein properties. Because of the differences in amino acid composition and sequence, and the possible presence of non-protein groups, each protein has different chemical characteristics that make it unique. These characteristics include size (molecular weight), charge, solubility, hydrophobicity and biological affinity. Differences in these characteristics are the basis for separation methods such as filtration, salt precipitation, and chromatographic procedures. A wide variety of separation methods are available. Strategies for Purification: A purification strategy includes a series of separation methods or steps. Each purification strategy is unique to the individual protein. This situation is in marked contrast to that for purification of DNA and RNA where only a few accepted purification protocols exist. This laboratory manual outlines the procedures involved in ONE strategy for protein purification. The choice of separation procedures for a particular protein purification strategy is partially based on the characteristics of the target protein that you want to purify. The choice of methods is also affected by the characteristics of the source material (microorganism, animal or plant tissue) from which the protein is being purified as well as the scale necessary (µg to kg amounts). The ideal purification strategy has the following goals: maximum recovery of the target protein; minimal loss of biological activity; and maximum removal of contaminating proteins, as well as low cost, as diagrammed in Figure 1.1. Like most protein purification strategies, purification of ß-galactosidase involves several protein separation steps along with procedures for analysis of purity and yield. The separation methods for this purification of ß-galactosidase will be outlined in Chapter 4.
Analysis of yield and purityIn order to purify a protein, it is essential to have a specific assay, a method for detecting and measuring the target protein. The assay makes it possible to determine the yield, which is how much of the target protein has been purified. In rare cases, the target protein can be detected easily because it is colored (myoglobin) or fluorescent (Green Fluorescent Protein) but most proteins are colorless. If the protein is an enzyme, such as ß-galactosidase, it may be assayed by its ability to catalyze a reaction, provided that there is a way to monitor the formation of product, or the disappearance of substrate. Enzyme These assays are often colorimetric assays based on a component of the reaction that absorbs light at a given wavelength. This light absorbance is measured by a spectrophotometer (See Seidman and Moore, Chapter 19 and 20 for more detailed treatment of spectrophotometry.) If the protein is not an enzyme, the assay is usually based on the biological activity of the protein. For example, if the target protein causes contraction of cultured muscle cells, an assay can be designed to measure this property. Since this specific assay for target protein is a measure only of the amount of target protein present and says nothing of the presence of contaminating proteins, an additional assay is needed to determine the amount of all proteins, called the total protein assay. The specific activity is expressed in units/mg and is a ratio of the amount of target protein (units) to the total mass of all proteins (mg). After each separation step is performed, an aliquot of the product mixture is set aside for analysis of purity and yield. As the purification process proceeds, the specific activity should increase because contaminants are being removed, thereby decreasing the total weight of all proteins in the sample. This indicates greater purity due to fewer contaminating proteins. Maximum specific activity will be obtained when the protein is purified to homogeneity; in other words, there are no other proteins present other than the target protein. Losses of yield are inevitable as the product mixture is manipulated - the goal is to minimize this loss. The assays used for measuring the activity of ß-galactosidase and monitoring the specific activity of this purification strategy will be described in more detail in Chapter 3.
But how do we know when we have removed ALL contaminants? Gel electrophoresis is one of the most powerful methods for analysis of protein purity. Protein samples are applied to a gel matrix and separated by size (SDS-PAGE), then stained to allow visualization of all proteins so the number of different proteins in the mixture can be determined. Two-dimensional PAGE offers even better resolving power since it separates by charge in the first dimension and size in the second dimension. Western blotting (immunoblotting) using specific antibodies to the target protein may also be used to verify that the target protein has been purified. Other methods, such as High Performance Liquid Chromatography (HPLC), may also be used to monitor purity but the instrumentation required is generally too expensive for teaching laboratories. Analysis and verification will be discussed in Chapter 5. Purification goals will vary according to application. The required purity of the end product will vary according to the intended use of the product. If complete removal of all contaminating proteins is not necessary, then it would not make good economic sense to spend time and money removing them. For example, a drug for human injection would obviously demand the highest standard of purity, even at high cost. But if the product is an enzyme for cutting DNA, a restriction endonuclease, then it might only be necessary to remove contaminating nucleases and other enzymes. The presence of some contaminating proteins would not affect product performance –in other words, the enzyme would be “functionally pure”. The scale of the purification process will affect the design of the purification strategy. Different purification strategies may be required in order to purify the same protein for two different applications. For example, if a researcher is trying to purify a very small amount of a protein in order to determine the sequence of amino acids in a particular protein, the purification strategy may be quite different from one required for the industrial production of the same protein for use as a drug. In both cases the protein must be very pure; however, the amounts needed vary from µg to kg so that a different purification strategy will probably be required. For these reasons, the exact approach that you will learn for ß-galactosidase in this lab manual will probably not work for another protein. Furthermore, the method which is presented in this lab manual is not the only method for purifying ß-galactosidase -- there are other methods which are equally effective. However, the concepts and techniques you will learn in this course will help you to understand and implement other strategies for protein purification that you will encounter. Throughout our purification project, we will discuss our approach for purifying and analyzing ß-galactosidase and compare it to alternative strategies of protein purification that could be used. Keep in mind, however, that it will not be possible to cover every possible strategy for protein purification in this course. Characteristics of the target protein, ß-galactosidaseß-galactosidase is an enzyme that is produced in E. coli. In the cell, ß-galactosidase hydrolyzes (splits) the ß 1,6 bond in lactose, a disaccharide, producing two monosaccharides, glucose and galactose, which can be used as energy sources. (See Figure 3.1) If lactose is unavailable, or the organism already has plenty of glucose as an energy source, then it would be inefficient for the cell to make ß-galactosidase. But if its substrate, lactose, is present, and glucose is low, then the enzyme is made (“induced”) because the cell needs it. Scientists use the term “inducible” to refer to enzymes that are only made when the inducing agent, usually the substrate of the enzyme reaction, is present. If an enzyme is made all the time, it is called “constitutive”. As a source material for purification of the enzyme, a mutant strain of E. coli that is constitutive for ß-galactosidase was chosen because this strain is producing the maximum amount of the target protein. Assignment: ß-galactosidase has been extensively studied and characterized. Commercial sources are available as we are certainly not the first to purify it.! Find ß-galactosidase in the Sigma catalog. How many different products are there? Look at the reported specific activity for each one. What accounts for these differences? Try typing ß-galactosidase into a search engine or a biomedical library database. (You will get thousands of hits.) From your research and discussion in lecture, what are some of the characteristics of ß-galactosidase? Overview of the Project We will begin the purification with cell paste from a strain of E. Coli that is a constitutive producer of ß-galactosidase and use the following series of procedures to purify and characterize this enzyme:
This manual is not designed to provide a complete body of theory for protein purification, although we do try to provide enough background information to understand the scientific basis of the methods in THIS purification strategy. Consult Appendix C for additional resources which present a more complete treatment of all separation methods. Classroom compromises: In the “real world”, the entire purification process outlined in this manual would routinely be performed in a cold room at 4°C, working quickly to minimize the loss of enzyme activity. Most classroom laboratories do not have access to cold rooms and working “quickly” is not possible in a class with 6 hours of laboratory time each week for 16 weeks. Fortunately, the activity of ß-galactosidase stands up admirably under these adverse conditions and most student groups still have activity to assay at the end of the semester. Important Tips for Protein PurificationKeep your protein cold. Throughout this project, the enzyme extract must be kept cold, in an ice bath or in a cold room or refrigerator. There are enzymes called proteases in the cell extract that become more active at temperatures above 4° C. These enzymes will hydrolyze other proteins including ß-galactosidase. So to keep the proteases inactive, and ß-galactosidase active, keep the extract cold. Use refrigerated buffers, refrigerated centrifuges (if possible) and keep your samples on ice at all times. Save materials. Never throw away any supernatant, pellet, fraction, or sample until you have completed analysis of that purification step. Use fume hoods for buffers. ß-mercaptoethanol (ß-ME) and dithiothreitol (DTT) are reducing agents that you will be adding to your buffers to preserve the activity of ß-galactosidase. DTT and ß-ME are volatile and gradually evaporate from solutions so you will be adding these compounds to your solutions just before you use them. They are extremely foul smelling and toxic at high concentrations. Use fume hoods to add the concentrated DTT to your working solutions and minimize the amount of time you have uncovered solutions at the bench. Keep Good Records. As always, it is extremely important that you keep extensive and accurate notes in your lab notebook. Follow all rules for keeping a notebook, such as using only indelible ink, never erasing anything, dating each entry and initialing your entries when appropriate. Keep track of the volumes you have at each step and other details such as where things are stored (which refrigerator or freezer, etc). Finally, for every chemical or solution, find and record the chemical name, concentration, supplier and catalog number and any other information you may need to reproduce your work. Remember, you - or another person unfamiliar with the work - should be able to repeat each step based on your laboratory notebook! Course OrganizationThe set of procedures in this manual is a project that will take most of the semester. Unlike many other classroom laboratory projects and experiments, the organization of this course is designed to model "the real world" where laboratory work is generally project based. The course is intentionally organized to give you an opportunity to work in partners or teams, fairly independently of the instructors. Presentation of Results. One of the most important aspects of biotechnology research is presentation of the results. Regardless of where you work, you will need to present your results orally and in written form to your supervisor and to others in the company or institution in which you are employed. It is also possible that your work will be presented at scientific meetings and, ultimately, be published in journals where other scientists will evaluate them critically. Each student group will present a final oral report to the class and each student will submit a written formal lab report on the purification project. Although this report is due when the project is completed, take the time now and several times during the first month of the semester to read over the guidelines for your report that are contained in Section 5 of this lab manual. Familiarity with this format will help you to collect and analyze your data in ways that will save you time and much grief when you write your report.
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