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PowerPoint Lectures for Introduction to Biotechnology, Second Edition William J.Thieman and Michael A.Palladino Chapter 4 Proteins as Products ---Monoclonal antibody as an example(補充) 吳輔祐教授 生物技術與動物科學系 Lectures by Lara Dowland Copyright © 2009 Pearson Education, Inc. Chapter Contents • • • • • • • • • • • 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 補充 Introduction to Proteins as Biotech Products Proteins as Biotechnology Products Protein Structures Protein Production Protein Purification Methods Verification Preserving Proteins Scale-Up of Protein Purification Postpurification Analysis Methods Proteomics monoclonal antibody--魔術子彈 字顏色代表---原來slide重點; 補充; 單株抗體 Copyright © 2009 Pearson Education, Inc. 4.1 Introduction to Proteins as Biotech Products • Proteins – large molecules that are required for the structure, function, and regulation of living cells • 2000 NIH launched Protein Structure Initiative – Effort to identify the structure of human proteins • 人體含>20,000種蛋白質。 • 一個細胞含>3,000種蛋白質。 Copyright © 2009 Pearson Education, Inc. 4.2 Proteins as Biotechnology Products(3-1) • Use of proteins in manufacturing is a time-tested technology – Beer brewing and winemaking – Cheese making • Recombinant DNA technology made it possible to produce specific proteins on demand – Enzymes – proteins that speed up chemical reactions – Hormones – Antibodies (修飾抗體結構--擬人化、親和力) Copyright © 2009 Pearson Education, Inc. 4.2 Proteins as Biotechnology Products(3-2) • Making a Biotech Drug – Produced through microbial fermentation or mammalian cell culture 細胞培養或小鼠腹腔製造單源抗體 – Complicated and time-consuming process – Must strictly comply with FDA regulations at all stages of the procedure Copyright © 2009 Pearson Education, Inc. 4.2 Proteins as Biotechnology Products(3-3) • Applications of Proteins in Industry – – – – – – – Medical applications--生長激素、單株抗體 Food processing Textiles and leather goods Detergents Paper manufacturing and recycling Adhesives: natural glues Bioremediation: treating pollution with proteins Copyright © 2009 Pearson Education, Inc. 4.3 Protein Structures(6-1) • Proteins – Are complex molecules built of chains of amino acids – Have electrical charge that causes them to interact with other atoms and molecules • Hydrophilic – water loving 親水性 • Hydrophobic – water hating 疏水性 Copyright © 2009 Pearson Education, Inc. 4.3 Protein Structures(6-2) • Structural Arrangement – four levels – Primary structure is the sequence in which amino acids are linked together 一級胺基酸序列 – Secondary structure occurs when chains of amino acids fold or twist at specific points • Alpha helices and beta sheets 二級α螺旋和β平板 – Tertiary structures are formed when secondary structures combine and are bound together 三級立體 – Quaternary structures are unique, globular, threedimensional complexes built of several polypeptides 四級複合體 Copyright © 2009 Pearson Education, Inc. 4.3 Protein Structures(6-3) Copyright © 2009 Pearson Education, Inc. 4.3 Protein Structures(6-4) • Protein Folding – The structure and function of a protein depends on protein folding – If protein is folded incorrectly, desired function of a protein is lost and a misfolded protein can be detrimental – 1951 two regular structures were described • Alpha helices and beta sheets • Structures are fragile; hydrogen bonds are easily broken Copyright © 2009 Pearson Education, Inc. 4.3 Protein Structures(6-5) • Glycosylation – post-translational modification wherein carbohydrate units are added to specific locations on proteins 抗體亦有糖化,可用以連接至其他分子。 • More than 100 post-translational modifications occur Copyright © 2009 Pearson Education, Inc. 4.3 Protein Structures(6-6) • Protein Engineering – Introducing specific, predefined alterations in the amino acid sequence through a process known as directed molecular evolution technology – Creating entirely new protein molecules 改變DNA序列以修改胺基酸序列,創造各種 新蛋白質。 Copyright © 2009 Pearson Education, Inc. 4.4 Protein Production • Proteins are valuable • Proteins are complex and fragile products • Production of proteins is a long and painstaking process – Upstream processing includes the actual expression of the protein in the cell – Downstream processing involves purification of the protein and verification of the function; a stable means of preserving the protein is also required Copyright © 2009 Pearson Education, Inc. 4.4 Protein Production • Protein Expression: The First Phase in Protein Processing – Selecting the cell to be used as a protein source • • • • Microorganisms (原核細胞)--inclusion body Fungi, yeast (真核細胞)--糖化 Plants Mammalian cell systems B cell與瘤細胞融合,產製單株抗體 • Whole-animal production systems (羊乳腺) • Insect systems Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-1) • Protein Must Be Harvested – Entire cell is harvested if protein is intracellular • Requires cell lysis to release the protein • Releases the entire contents of the cell – Culture medium is collected if the protein is extracellular Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-2) • Separating the Components in the Extract – Similarities between proteins allow the separation of proteins from non-protein material • Protein precipitation – salts cause proteins to settle out of solution • Filtration (size-based) separation methods – Centrifugation – Membrane filtration – Microfiltration – Ultrafiltration Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-3) • Separating the Components in the Extract • Diafiltration and dialysis rely on the chemical concept of equilibrium Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-4) • Separating the Components in the Extract – Differences in proteins allows the separation of the target protein from other proteins • Chromatography – allows the sorting of proteins based on size or by how they cling to or dissolve in various substances Copyright © 2009 Pearson Education, Inc. 色層分析(Chromatography) (14-5) • • • • 分子大小:gel filtration 正負電:cation, anion exchange 等電點:isoelectric focusing 疏水性:hydrophobic interaction reverse phase HPLC • 親和性:affinity (抗體、His tag-Ni2+) • 設備:LC(低壓大量)、FPLC、HPLC(高壓微量) Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-6) • Separating the Components in the Extract – Chromatography • Size exclusion chromatography (SEC) – uses gel beads with pores – Larger proteins move quickly around the beads and smaller proteins slip through the pores and therefore move more slowly through the beads – 管柱細長 Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-7) Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-8) • Separating the Components in the Extract – Chromatography • Ion exchange chromatography – relies on the charge of the protein – Resin is charged – Opposite charged proteins will stick to resin beads – Can be eluted by changing the charge with salts of increasing concentration – 管柱粗短 Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-9) Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-10) • Separating the Components in the Extract – Chromatography • Affinity chromatography relies on the ability of proteins to bind specifically and reversibly to uniquely shaped compounds called ligands • 單株抗體連結至resin • 重組蛋白接6 His tag,以Ni2+分離 Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-11) • Separating the Components in the Extract – Chromatography • Hydrophobic interaction chromatography (HIC) sorts proteins on the basis of their repulsion of water Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-12) Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-13) • Separating the Components in the Extract – Iso-electric focusing used in QC to identify two similar proteins that are difficult to separate by any other means • Each protein has a specific number of charged amino acids on its surface in specific places • Creates a unique electric signature known as its iso-electric point (IEP) where charges on the protein match the pH of the solution Copyright © 2009 Pearson Education, Inc. Bio-Rad Rotofor Cell 28 Copyright © 2009 Pearson Education, Inc. 4.5 Protein Purification Methods(14-14) • Separating the Components in the Extract – Analytic methods • High-Performance liquid chromatography (HPLC) – uses high pressure to force the extract through the column in a shorter time • Mass spectrometry (mass spec) – highly sensitive method used to detect trace elements – Used to indicate the size and identity of most protein fragments Copyright © 2009 Pearson Education, Inc. 4.6 Verification • The presence and concentration of the protein of interest must be verified at each step of the purification process – SDS-PAGE (polyacrylamide gel electrophoresis) (Fig 4.15) – Western blotting – ELISA--酵素免疫分析,以抗體偵測 Copyright © 2009 Pearson Education, Inc. 4.7 Preserving Proteins • Lyophilization (freeze-drying) – Protein, usually a liquid product, is first frozen – A vacuum is used to hasten the evaporation of water from the fluid – Will maintain protein structure and can be stored at room temperature for long periods of time Copyright © 2009 Pearson Education, Inc. 4.8 Scale-Up of Protein Purification • Protocols are usually designed in the laboratory on a small scale • Must be scaled up for production – Process is approved by FDA so must make sure laboratory procedures can be scaled up Copyright © 2009 Pearson Education, Inc. 4.9 Postpurification Analysis Methods • Protein Sequencing – Must determine the primary structure, the sequence of amino acids • X-ray Crystallography – Used to determine the complex tertiary and quaternary structures. 分子必須形成結晶。 NMR (核磁共振)--分析較複雜。 Copyright © 2009 Pearson Education, Inc. 4.10 Proteomics • A new scientific discipline dedicated to understanding the complex relationship of disease and protein expression – Uses protein microarrays to test variation in protein expression between healthy and disease states • 各跑2D電泳,比對兩者間的差異,取出定序。 Copyright © 2009 Pearson Education, Inc. 補充: monoclonal antibody製造 • 免疫小鼠,取血清為polyclonal antibody。特異性較低 ,不可重複製造。 • 取免疫小鼠之脾細胞,與瘤細胞融合,經篩選得不死 的細胞,能分泌單株抗體。特異性高,可重複大量製 造,特性完全相同。 Copyright © 2009 Pearson Education, Inc. 36 Copyright © 2009 Pearson Education, Inc. 台大莊榮輝教授網站:http://juang.bst.ntu.edu.tw/ECX/monoclonal.htm 37 Copyright © 2009 Pearson Education, Inc. 單源抗體的應用 • • • • • 酵素免疫分析---ELISA, Western blot 親和性管柱或磁珠---快速分離濃縮 flow cytometry immunohistochemistry therapeutic---magic bullet 接藥物或ADCC(Ab dependent cell-mediatedd cytotoxicity)殺死癌細胞 中和細胞激素,如Denosumab中和RANKL,抑制 osteoclast成熟,半年打一針防骨質疏鬆 目前在臨床測試的生物醫藥>30%為單株抗體,市 場>300億美金/年 38 Copyright © 2009 Pearson Education, Inc.