Download 4.5 Protein Purification Methods

PowerPoint Lectures for
Introduction to Biotechnology, Second Edition
William J.Thieman and Michael A.Palladino
Chapter 4
Proteins as Products
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
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
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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
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4.2 Proteins as Biotechnology Products
• 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
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4.2 Proteins as Biotechnology Products
• Making a Biotech Drug
– Produced through microbial fermentation or mammalian
cell culture
– 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
• Applications of Proteins in Industry
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Medical applications
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
• 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
• Hydrophobic – water hating
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4.3 Protein Structures
• Structural Arrangement – four levels
– Primary structure is the sequence in which amino acids are
linked together
– Secondary structure occurs when chains of amino acids fold
or twist at specific points
• Alpha helices and beta sheets
– Tertiary structures are formed when secondary structures
combine and are bound together
– Quaternary structures are unique, globular, threedimensional complexes built of several polypeptides
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4.3 Protein Structures
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4.3 Protein Structures
• 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
• Glycosylation – post-translational modification
wherein carbohydrate units are added to specific
locations on proteins
• More than 100 post-translational modifications
occur
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4.3 Protein Structures
• 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
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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
Fungi
Plants
Mammalian cell systems
Whole-animal production systems
Insect systems
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4.5 Protein Purification Methods
• 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
• 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
• 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
• 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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4.5 Protein Purification Methods
• 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
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4.5 Protein Purification Methods
Copyright © 2009 Pearson Education, Inc.
4.5 Protein Purification Methods
• 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
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4.5 Protein Purification Methods
Copyright © 2009 Pearson Education, Inc.
4.5 Protein Purification Methods
• 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
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4.5 Protein Purification Methods
• 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
Copyright © 2009 Pearson Education, Inc.
4.5 Protein Purification Methods
• 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.
4.5 Protein Purification Methods
• 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)
– Western blotting
– ELISA
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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
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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
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