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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
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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
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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。
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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)
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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
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4.2 Proteins as Biotechnology Products(3-3)
• Applications of Proteins in Industry
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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
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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
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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
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4.3 Protein Structures(6-3)
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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
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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
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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。
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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
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4.4 Protein Production
• Protein Expression: The First Phase in Protein
Processing
– Selecting the cell to be used as a protein source
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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
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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
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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
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4.5 Protein Purification Methods(14-3)
• Separating the Components in the Extract
• Diafiltration and dialysis rely on the chemical concept of
equilibrium
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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
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色層分析(Chromatography) (14-5)
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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)
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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
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4.5 Protein Purification Methods(14-7)
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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
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4.5 Protein Purification Methods(14-9)
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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
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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
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4.5 Protein Purification Methods(14-12)
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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
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Bio-Rad
Rotofor Cell
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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
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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
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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
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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
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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。
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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。
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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。
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台大莊榮輝教授網站：http://juang.bst.ntu.edu.tw/ECX/monoclonal.htm
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單源抗體的應用
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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
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