Copyright © 2009 Pearson Education, Inc. PowerPoint Lectures for Introduction to Biotechnology, Second Edition William J.Thieman and Michael A.Palladino Lectures by Lara Dowland 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

Copyright © 2009 Pearson Education, Inc. Chapter Contents 4.1 Introduction to Proteins as Biotech Products 4.2 Proteins as Biotechnology Products 4.3 Protein Structures 4.4 Protein Production 4.5 Protein Purification Methods 4.6 Verification 4.7 Preserving Proteins 4.8 Scale-Up of Protein Purification 4.9 Postpurification Analysis Methods 4.10 Proteomics 補充 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, three- dimensional 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-Ni 2+ ) 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 Ni 2+ 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. 28 Bio-Rad Rotofor Cell

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 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： 37

Copyright © 2009 Pearson Education, Inc. 38 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