Transcript Introduction to Biotechnology Chapter 4: Proteins as Products Fall 2006

Introduction to Biotechnology
Chapter 4: Proteins as
Products
Fall 2006
Examples of Proteins Purified

Enzymes
– Depolymerizases
• Amylases, proteases, lipases



Hormones
Antibodies
What was the first recombinant protein to be mass
produced?
– Human replacement proteins – used to treat a disease
that is caused by a gene that fails to produce a
necessary protein or that produces a dysfunctional
protein
More Examples:
Food Processing (the creamy in ice cream)
 Textiles and Leather Goods (bio-stoning)
 Detergents (enzymes)
 Paper Manufacturing and Recycling
 Adhesives: Natural Glues
 Bioremediation: Treating Pollution with
Proteins (metallothioneins)

Protein Structure, Function,
and Analysis
Replication
Transcription
Translation
A
B
B
Active Site
A
A B
Coding: 5’-GATCTGAATCGCTATGGC-3’
Template: 3’-CTAGACTTAGCGATACCG-5’
mRNA:
mRNA 5’-GAUCUGAAUCGCUAUGGC-3’
tRNA:
CUAGACUUAGCGAUACCG
amino acid:
asp. acid, leucine, asparag, arginin, leucin, glycine
Biochemical Differences
• Changes in DNA
proteins with:
– different functions
– novel traits
– positive, negative or no effects
• Genetic diversity provides pool for natural
selection = evolution
Biochemical Similarities
• Traits are the result of:
– Structure
– Function
• Proteins determine structure and function
• DNA codes for proteins that confer traits
DNA
RNA
Protein Trait
Post-Translational Modifications
Glycosylation- carbohydrate units added to
proteins
Occurs in the Golgi
Function- increase solubility, orient proteins
in membranes, extend the life of proteins
Protein Production

Upstream Processing: the actual expression of the
protein in the cell
– Microorganisms- cheap, well understood, grow rapidly,
produce large amounts, clone in as cDNA, fusion gene
(fusion protein), inclusion bodies, no glycosylation
(review table 4.3)
– Fungi – can do some posttranslational modifications
– Plants- 85% of current drugs from plants; rapid growth,
cheap, proteins not expressed properly
– Mammalian Cell Systems – finicky, grow slowly, and
expensive, BUT processes human proteins correctly
• TPA – first drug produced by mammalian cell culture
– Whole-animal –transgenic (goats making spider silk)
– Insect systems – baculoviruses are used as vector to
insert genes into insect cells
Scale-up of Protein Purification
R&D starts with a small-scale level
 Production may demand a larger level

– Small scale may not be adaptable
– If FDA approval has been gained for smallscale, cannot change the parameters when
scaled up (so scientists MUST make sure they
can scale up before seeking approval)

Downstream Processing: the protein is
separated from other parts of the cell and
then isolated from other proteins
– Preparing the Extract for Purification
• If intracellular, lyse the cells
– Stabilizing the Proteins in Solution
• Temperature, decrease protease activity and
denaturing activity, maintain biological activity
– Separating the Components in the Extract
• Utilize the chemical and physical properties of
proteins to separate them

Protein precipitation
– Ammonium sulfate
Centrifugation (sized based)
 Filtration

– Membrane, microfiltration, ultrafiltration
– Diafiltration and dialysis

Chromatography
– Size-exclusion, ion-exchange, affinity,
hydrophobic, iso-electric focusing, 2D
electrophoresis

Analytic Methods
– HPLC, mass spectrometry
Column Chromatography
Separates proteins by size, charge, or chemical properties
depending on matrix of column and chemical differences of
proteins to be separated.
• Resin – solid particles in the column
• Matrix – resin, or solid part of the column which has pores
that allow the molecules to pass.
• Sample – mixture which is loaded
onto the column to be separated
• Elution- liquid that passes out of
the column (is eluted) and can be
collected
• Fraction – a portion of the elution
which is collected separately
**Important note – If your sample is
too concentrated, you may overload
the column, which means there are not enough binding sites
in the column for all of the pigment molecules and some
that should stay in the column will instead wash through.
• Gel-filtration or size exclusion chromatography
Separates molecules based on size
Larger molecules pass quickly around the beads and
drain, or elute, from the column faster because smaller
molecules spend more time in the beads, or resin.
• Ion-exchange chromatography
Protein of interest attaches to matrix because of its charge
and all other proteins wash through the column
A new buffer with a greater charge is washed through
which knocks the protein off the matrix and takes its
place
• Hydrophobic interaction chromatography
 A hydrophobic (“water-fearing” or nonpolar) buffer is added to the
column and the sample.
 This makes the protein hydrophobic because it drives the hydrophilic
(“water-loving” or polar) amino acids to the center of the molecule
 The now hydrophobic protein attaches to the hydrophobic column matrix
 Wash buffers of increasing hydrophilicity are added to the column,
and proteins will lose their affinity for the hydrophobic column and
wash off.
 Water (the most hydrophilic liquid) washes all remaining proteins
off of the column.
Verification

SDS-PAGE review figure 4.14
SDS-Polyacrylamide Gel
Electrophoresis (SDS-PAGE)
• SDS (Sodium Dodecyl Sulfate) detergent
–solubilizes and denatures
proteins
–negative charge to proteins
CH3
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
• Heat denatures proteins
CH2
O
O S O
-
O
SDS
Why Use Acrylamide Gels to
Separate Proteins?
• Acrylamide gel
tight matrix
• Ideal for protein separation
• Smaller pore size than agarose
• Proteins much smaller than DNA
– average amino acid = 110 Da
– average nucleotide pair = 649 Da
– 1 kilobase of DNA = 650 kDa
– 1 kilobase of DNA encodes 333 amino acids = 36 kDa
How does an SDS-PAGE gel
work?
s-s
SDS, heat
proteins with
SDS
-
+
Lane
1. Kaleidoscope Markers
2. Shark
3. Salmon
4. Trout
5. Catfish
6. Sturgeon
7. Actin and Myosin Standard
Preserving Proteins

Lyophilization (freeze drying)
– First frozen, placed under vacuum to hasten the
evaporation of water (I.e. ice crystals go to
water vapor). The containers are sealed after the
water is removed, leaving the dried proteins
behind.
Post-Purification Analysis
Methods
Protein Sequencing
 X-ray Crystallography

Proteomics

Proteomes are compared under healthy and
diseased states
– The variations of protein expression are then
correlated to onset or progression of a specific
disease

Protein chips
– Biochips that can be used to identify proteins
(figure 4.15)