Chapter 5

Chapter 5 - Chapter 5: Protein Purification 1. Purify known...

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Chapter 5: Protein Purification 1. Purify known proteins a. Example: working with RNA Polymerase to adapt it for pharmaceuticals 2. Purify unknown proteins involved in a cellular process I. Characteristics of Proteins a. Every protein has a unique primary sequence. Hence, every protein has several unique characteristics. i. Size (molecular weight) of the protein ii. Charges of proteins based on the types of the amino acids in the protein 1. Basic (pI above 7), Acidic (pI below 7) Proteins iii. Subunit compositions 1. Ex. Some proteins have one subunit, others have more iv. Solubility 1. Due to the types of amino acids that make up the protein v. Natural Binding Affinity 1. Some have a natural ability to bind certain ligands a. Example. All eukaryotic mRNA have a poly-AAA tail. Back in the day when people where interested in what binds, they made a resin that had nothing but a binding to it and then they discovered that poly-AAA tail b. As biochemists, we are interested in understanding the chemical and biological properties of proteins. Consequently, we need methods to selectively isolate the proteins of interest from the rest of the cell i. Approximate number of proteins in a E. Coli cell is about 3,000-4,000 ii. Approximate number of proteins in a human cell is about 25,000-30,000 genes in the cell that can code proteins II. Precautions a. Factors that influence protein stability. We are taking a protein from its intercellular environment, which is a very density packed place. There are a lot of marcomolecules crowding the cell and these play a role in keeping proteins in there proper state. When you take the proteins about of the cell, you are placing them into a dilute environment i. Temperature 1. Whenever proteins are purified the temp is mostly keep less than 10 degrees C. ii. pH iii. Ionic Strength
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1. The same thing as salt concentration III. Methods of cell disruption a. There are a number of ways to prepare a protein extract from prokaryotic and eukaryotic cells. i. Mechanical methods 1. Blenders 2. Sonication a. Uses high frequency sound waves which actually break cells down – it is a very effective method 3. French Press – modification ii. Enzymatic methods 1. Lysozyme: enzyme that degrades bacterial cell walls b. Maintain the stability of proteins once cells have been lysed i. Be sure to keep your proteins happy 1. Formulate a buffer to mimic the intracellular pH and the ionic strength a. If you have a cell that has a pH of 7 and a with NaCl, you are going to make the buffer the same c. Monitoring for the presence of a proteins i. Enzyme activity assay 1. Alkaline phosphatase a. PNPP in vitro which is clear but in the presence of alkaline phosphatase it is yellow and has a maximum absorbance of 410 nm b. If you don’t know what your looking for this is very difficult to do IV. Protein Purification Methods a. Solubility, which is usually the first method employed
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Chapter 5 - Chapter 5: Protein Purification 1. Purify known...

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