Online Counseling Resource YCMOU ELearning Drive… School of Architecture, Science and Technology Yashwantrao Chavan Maharashtra Open University, Nashik – , India

OC-SEP-SBI084-CP01-01 Introduction Programmes and Courses  SEP-SBI084-U01-CP01

School of Science and Technology, Online Counseling Resource… Credits  Academic Inputs by Sonali Alkari Faculty YCMOU Nagpur Centre, Faculty LAD college P.G. D of Biotechnology Research officer Ankur Seeds Pvt Ltd

School of Science and Technology, Online Counseling Resource… © 2008, YCMOU. All Rights Reserved.4 How to Use This Resource  Counselor at each study center should use this presentation to deliver lecture of minutes during Face-To-Face counseling.  Discussion about students difficulties or tutorial with assignments should follow the lecture for about minutes.  Handouts (with 6 slides on each A4 size page) of this presentation should be provided to each student.  Each student should discuss on the discussion forum all the terms which could not be understood. This will improve his writing skills and enhance knowledge level about topics, which shall be immensely useful for end exam.  Appear several times, for all the Self-Tests, available for this course.  Student can use handouts for last minutes preparation just before end exam.

School of Science and Technology, Online Counseling Resource… © 2007, YCMOU. All Rights Reserved.5 Learning Objectives After studying this module, you should be able to: Explain purpose of protein purification Explain basis of protein purification Explain methods of protein purification.

School of Science and Technology, Online Counseling Resource… Introduction  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.  During the last decade, the genomic sequence of many organisms has been determined.  The wealth of information in these genomes has dramatically changed research. © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Protein Purification  The development of techniques and methods for protein purification has been as essential prerequisite for many of the advancements made in biotechnology.  Protein purification varies from simple one step precipitation procedure to large scale validated production processes.  Often more than one purification step is necessary to reach desired purity.  The key to successful and efficient purification is to select the most appropriate techniques, optimize their performance to suit the requirements and combine them in logical way to maximize yield and minimize the number of step required. © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Need for Protein Purification :1  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 for any of the following reasons:  The purified protein can be used to determine the amino acid sequence. © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Need for Protein Purification:2  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. © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Need for Protein Purification :3  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).  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. © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Need for Protein Purification :4  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.)  Purification procedures attempt to maintain the protein in native form. Although some proteins can be re-natured, most cannot! © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Basics of Protein Purification:1 Property Methods solubilityPrecipitation with ammonium sulfate (salting out)* Size / shapeSize-exclusion chromotography Isoelectricpoint (charge)Ion exhange chromatography binding to small moleculesAffinity chromatography © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Basics of Protein Purification:2  A protein must be purifies before its structure and the mechanism of its action can be studied.  However, because protein vary in shape, size, charge, and water soulbility, no single method can be used to isolate protein.  To isolate one particular protein from the estimated 10,000 different proteins in a cell as a daunting task that requires methods both for separating proteins and detecting the presence of proteins. © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Basics of Protein Purification:3  Any molecule, whether protein, carbohydrate or nucleic acid, can be separated from other molecule based on large differences in some physical characteristics.  Although the sequence of amino acid in a protein uniquely determines its function, the most useful physical characteristics for separation of protein is size, defined as either length or mass.  To purify a protein from a mixture, biochemists exploit the ways that individual proteins differ from one another.  They differ in: physical properties such as their size, shape, mass, charge solubility and Thermal stability.

School of Science and Technology, Online Counseling Resource… Methods of Purification:1  Separation methods based on simple physical properties, from a very significant aspect of research in biology.  The isolation of molecule from biological material in the pure form, is a prime prerequisite for understanding the nature and function of these compounds.  This involves the separation of one molecular species from mixture of compounds.  This involves the separation of one molecular species from mixture of compounds closely resembling each other properties. © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Methods of Purification:2  The chemical methods of organic analysis employ extremes of pH/temperature, organic solvent or oxidizing /reducing agents which alter or destroy the structure of many biological molecules.  But the most of the methods of separating molecules involve technique utilizing the differences in physical properties such as their size, shape, mass, charge solubility. © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Protein Purification:Making Protein Extract 1. Extract proteins from cell 2. Separate extract contents by centrifugation to make protein extract 3.Separate protein extract by column chromatography - ion exchange - gel filtration - affinity chromatography 4. Analyze purity of isolated protein by gel electrophoresis © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Protein Purification:Making Protein Extract © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Break cells, tissue, or organ Blender, homogenize r, sonication, pressure, psmotic Pellet with intact cells, organelles, membranes and membrane proteins Supernatant with Soluble protein Preparing the Sample – Crude Extract Protein from cells or tissue © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Guidelines for Protein Purification  The guidelines for protein purification can be applied to any purification process  These guidelines provide a systematic approach to the development of an effective purification strategy.  These guidelines are as follows: Define objective of purity, activity and quantity required for final product. Define properties of target protein and critical impurities to simplify technique selection and optimization. © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… What You Learn… You have learnt : protein purification is an essential prerequisite for many of the advancements made in biotechnology. Differenent physical properties such as their size, shape, mass, charge solubility are used for separation. Technique involved in separation are centrifugation, chromatographic techniques such as ion exchange, gel filteration, affinity chromatography. © 2008, YCMOU. All Rights Reserved.

School of Science and Technology, Online Counseling Resource… Critical Thinking Questions 1.Why proteins are needed in pure form? 2.State the various parameters responsible for protein purification. 3.Describe the various techniques used in protein purification. © 2008, YCMOU. All Rights Reserved.22

School of Science and Technology, Online Counseling Resource… Hints For Critical Thinking Question 1.protein purification is an essential prerequisite for many of the advancements made in biotechnology. 2.size, shape, mass, charge solubility 3.centrifugation, chromatographic techniques © 2008, YCMOU. All Rights Reserved.23

School of Science and Technology, Online Counseling Resource… Study Tips:1  Book1 Title: Biophysical Chemistry (principles and techniques ) Author: Upadhay. Upadhay.Nath Publisher:Himalaya publishing House  Book2 Title: Physical Biochemistry (application to Biochemistry and molecular biology) Author: Freifelder Publisher: W. H. Freeman and Company

School of Science and Technology, Online Counseling Resource… Study Tips:2  Book3 Title: Essentials of Biophysics Author: Narayanan Publisher: New Age Int. Pub. New Delhi.  Book4 Title: A Text Book of Biophysics Author: Roy R.N. Publisher:New Central Book Agency

School of Science and Technology, Online Counseling Resource… Study Tips Microsoft Encarta Encyclopedia Wikipedia the free encyclopedia

School of Science and Technology, Online Counseling Resource… End of the Presentation Thank You !