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

• 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

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
– Goal is to be able to model unknown protein structures
based on structural comparisons to those stored in the
database
• More than 1,200 superfamilies of proteins identified so far
• Relationship between protein's amino acid sequence to its
structure is well understood
• Public database holds more than 33,000 protein sequences

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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

– 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, three-
dimensional 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
• This change can affect the protein's activity by
increasing:
– its solubility
– orient proteins into the membranes
– extend the active life of a molecule in an organism

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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
• Can induce mutations randomly into genes and then select the
bacterium with the protein product (enzyme) that has highest
activity (example: a company was able to produce bacteria and
industrial enzymes that tolerate a cyanide concentration that is
normally too high for most bacteria to survive)
– Resulting "selected" bacteria can be used to remediate cyanide
contamination resulting from mining/other waste accumulation

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 4.2 Protein Structures

• Protein Engineering
– Example using biotechnology to form synthetic infectious
protein particles to study the following diseases
• Faulty protein folding is expressed as a disease caused by
infectious protein particles called prions which attract normal cell
proteins and induce changes in their structure leading to
accumulation of useless proteins that actually damage cells
– Prion can occur in sheep and goats (scrapie) and cows (mad cow
disease)
– Human forms of these brain destroying diseases include kuru and
transformable spongiform encephalitis
– All of these diseases involve changes in conformation of the prion
precursor protein (protein normally found in mammalian neurons as
a membrane glycoprotein)

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 4.2 Protein Structures

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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
• Microorganisms
• Fungi
• Plants
• Mammalian cell systems
• Whole-animal production systems
• Insect systems

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 4.3 Protein Production

• Protein Expression: Upstream Processing

– There are pros and cons to expressing proteins in
bacteria
• Prokaryotic cells are unable to carry out processes such as
glycosylation
• Most common bacterial species used is E. coli
• Bacteria
– Fermentation processes are well understood
– Cultured in large quantities in short period of time
– Relatively easy to alter genetically

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 Protein Expression: Upstream
Processing
– Selecting the cell to be used as a protein source
• Bacteria- majority of proteins synthesized naturally by E coli are
intracellular
– Most cases resultant foreign protein accumulates in cell's cytoplasm
in form of insoluble clumps = inclusion bodies which must be
purified from other proteins
• Sometimes the genetically engineered E coli produce the desired
protein in the form of a fusion protein
– Target protein is fused to bacterial protein so an additional step is
necessary to break the two apart
» Fused bacterial protein is usually an enzyme that will bind to its
substrate and can be attached to a purification column

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 4.3 Protein Production
• Protein Expression: Upstream Processing
– Fungi
• Source of wide range of proteins used in products including
animal feed and beer
• Many species of fungi are used as hosts for engineered
proteins
• Fungi are eukaryotic and capable of posttranslational
modifications to allow proper folding of proteins

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 4.3 Protein Production

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 4.3 Protein Production

• Protein Expression: Upstream Processing

– Mammalian cell culture systems = challenging
• Nutritional requirements are complex
• Mammalian cells grow slowly
• Easy to contaminate
• Best choice for proteins destined to be used in humans

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 Protein Expression: Upstream
Processing
– Plants: used for protein expression
– Plants can be genetically engineered to produce specific
proteins that do not occur naturally
• Process encourages rapid growth and reproductive
rates in plants
– Tobacco plant- great choice for biotech protein
production
– Disadvantages using plants: not all proteins can be
expressed in plants; have cell wall which makes
purification difficult; process of glycosylation is slightly

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 4.3 Protein Production

• Protein Expression: Upstream Processing

– Animal bioreactor production systems
– Use for monoclonal antibody production
• Mice are injected with an antigen and mouse secretes the
desired antibody and the antibody is then purified
– What is an antibody? Proteins produced in reaction to
antigens- invading viruses or bacteria
• Antibodies combine with and neutralize an antigen protecting the
organism
• Production of antibodies is part of immune response that helps
living things resist infectious disease

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 4.3 Protein Production

• Protein Expression: Upstream Processing

– Insect system
• Baculoviruses (viruses that infect insects) are used as
vehicles to insert mammalian DNA causing the desired
proteins to be produced by insect cells
– Sometimes the posttranslational modification of
proteins is slightly different in insects than in mammals
– Currently used when small quantities of proteins are
needed in research

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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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 4.3 Protein production

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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

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 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

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 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

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 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

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 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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