Transcript Powerpoint SDS PAGE

Expression
Induction
pLATE31 Vector
pLATE Vector Landmarks
• T7 Promoter
• LacO
• RBS
• His*Tag coding sequence
• Multiple cloning sites
• lacI coding sequence
• bla(ApR) coding sequence
The Lac Operon
The Lac Operon is a “switch” that turns lactose specific
genes on and off
• An operon is a cluster of genes with related functions and
contains control sequences that turn the genes on or off
• Example: Lac operon in the bacterium E. coli uses:
– A promoter, a control sequence where the transcription enzyme initiates
transcription
– An operator, a DNA segment that acts as a switch that is turned on or off
– A repressor, which binds to the operator and physically blocks the
attachment of RNA polymerase
Lactose is broken down into two
simple sugars by the enzyme b-gal
b-gal is only present when glucose is not
present AND when lactose IS present.
How does this happen?
The Lac Operon Players
• LacI : gene for a repressor protein; it has its own promoter and terminator and is
always being made in the cell
• Operator: the DNA sequence to which the LacI repressor binds
• CAP protein: will bind to the Lac promoter only when glucose is absent; when it
does so, it helps RNA polymerase bind to the promoter and begin to make mRNA
• A single promotor controls transcription of all 3 genes at the same time. So, a
single mRNA is created that is translated into 3 different proteins.
• LacZ: the gene for betagalactosidase which cleaves lactose into simple
sugars
• LacY: the gene for permease which pumps more lactose into the cell
• LacA: the gene for transacetylase which modifies betagalactosidase for some
unknown reason
The Lac Operon
CAP protein
helps RNA
polymerase
bind the
promoter.
But CAP
protein will
only do this
when
glucose
levels are
low. This
helps RNA
polymerase
bind to the
promoter.
Repressor
binds to the
operator
unless
lactose is
present. If
lactose is
present, it
will bind to
the
repressor
and cause it
to release
from the
operator
sequence.
So, the only time you get the Lac operon genes to express is when there is low glucose AND lactose is
present. This ensures that genes needed for lactose metabolism are only produced when they are
needed.
How might we use the LacZ promotor
to express a recombinant protein?
We create a gene construct in which Your Favorite
Gene is under the control of the Lac promoter and
operator.
What happens to the lactose when we
add it to the media?
Shortly after adding lactose to the media, the Lac operon is activated both in
your plasmid and in the normal Lac operon genes in the cell. So for a short
time, YFG is expressed. But not long after, all the lactose is depleted because
the cell is also making the genes necessary to “eat” the lactose.
As a result, expression of YFG is reduced shortly after adding
lactose because the lactose is all converted into galactose and
glucose.
What is the point of using IPTG instead of
lactose to induce protein expression?
This stuff is close
enough to real
lactose that it will
bind to the lacI
repressor and
cause it to no
longer bind the
operator, but it is
different enough
that
betagalactosidase
won’t chop it up.
So we can use it
to give long term
induction of our
YFG from the lac
promoter!!!
Induction Procedure
• Prepare a starter culture: inoculate 3ml of LB +
ampicillin with a single colony
• Incubate at 37°C with shaking to an OD600 reading of
0.5 (~2.5 x 108 cells)
• Scale up: add 3ml culture to 100ml LB + ampicillin
• Incubate at 37°C with shaking to an OD600 reading of
0.5-1.0 (~2.5-5.0 x 108 cells)
• Take 10ml of starter culture and add IPTG to a final
concentration of 0.5mM
• Harvest cells at variable time points after induction:
–
–
–
–
0min
30min
1½ h
3-4 h
What do you expect you’ll see
at the different time points?
Protein Extraction
Where is my protein?
In the medium?
• Target protein leakage from cells
– Prolonged induction
• Target protein is exported
In the periplasm?
• Vector/target protein includes signal sequences
In the cytoplasm?
• Soluble
• Insoluble
How do I get to my protein?
If the target protein is within the cytoplasm,
there are two plasma membranes to get past:
• Mechanical disruption
– (i.e. sonication, grinding)
~OR~
• Chemical disruption:
– (i.e. detergents, lysozyme)
• Centrifuge
Where is my protein?
Lysis Buffer Components
• Buffer solution used for the purpose of lysing cells
• Possible components:
• Buffer:
• Protease inhibitors:
– (i.e. aprotinin, benzamidine, EDTA, Leupeptin, PMSF, Pepstatin A)
• Other additives:
– Salts: maintain ionic strength of the medium
– Detergents: breaks down plasma membranes, solubilizes poorly
soluble proteins
– Glycerol: stabilization of proteins
– Glucose/sucrose: stabilizes lysosymal membranes
– Reducing agents: reduces oxidation damage
– Ligands/metal ions: stabilization
Protein Purification Protocol
• https://www.youtube.com/watch?v=K8VFwhYLLm0
• After harvest, freeze the cells
• Thaw cells and resuspend in lysis buffer
– Tris-HCl + glycerol + NaCl
• Add lysozyme
• Incubate
• Centrifuge Where is my protein?
Where are all the other (soluble
cytoplasmic) cell proteins?
Tags and Affinity
Chromatography
What is affinity chromatography?
• A technique used to separate and purify a biological molecule from
a mixture based on the attraction of the molecule of interest to
a particular ligand which has been previously attached to
a solid, inert substance.
• The mixture is passed through a column containing the ligand attached to
the stationary substance so that the molecule of interest stays within
the column while the rest of the mixture continues through to
the through. Then, a different chemical is flushed through the column to
detach the molecule from the ligand and bring it out separately from
the rest of the mixture.
• Commonly used affinity columns:
– Ni2+  binds to Histidines (example 6xHis)
– Specific antibodies (anti-S tag)
– glutathione  binds to GST
– Protein A or G  binds antibodies
Ni-NTA columns are a type of affinity
chromatography
The high affinity of the Ni-NTA resins for 6xHistagged proteins or peptides is due to:
• the strength with which the Nickel ions are held to the
NTA molecule
• the specificity of the interaction between histidine
residues and immobilized nickel ions
NTA has a chelating group that
occupies four of six sites on the
nickel ion
Ni-NTA columns
This shows
two of the
6 histidines
in the His
tag that is
present on
the protein
(shown in
blue)
Here is the Nickel ion
that is bound between
the protein His tag and
the NTA molecule
(shown in black)
Here is the NTA
molecule (shown in
red) that is attached to
the solid support in the
column
The high affinity of the Ni-NTA resins for 6xHis-tagged proteins or peptides
is due to:
Protein elution
Elution of His tagged proteins can be achieved
either by reducing the pH or by competition with
imidazole.
What is an advantage of using competition with
imidazole?
• It is often preferred because it is so gentle.
Changing the pH or using denaturants can
damage the protein, but competition with
imidazole has no such adverse effects.
Why Imidazole?
Imidazole is part of the structure of
histidine that is responsible for binding
to Nickel. So using free imidazole at
high concentrations outcompetes the
binding of the histidine imidizole ring.
Ni
2+
Ni
2+
Ni-NTA columns
Since imidazole has the same ring
structure as histidine, it will bind
to the Ni atoms too. It is at very
high concentrations and so
displaces the HIS tag from the
protein…as a result, the protein
washes out of the column
SDS-PAGE
Why is gel electrophoresis more
challenging for proteins?
• The movement of any charged species
through an electric field is determined by:
– Net charge
– Size
– Magnitude of applied field
• What is the net charge of native proteins?
• Is the length of a protein always proportional
to its molecular radius?
SDS-PAGE
• Sodium Dodecyl Sulfate Polyacrylamide Gel
Electrophoresis
– Developed by Ulrich K. Laemmli (Nature 227: 690685, 1970)
• Separate proteins according to their
electrophoretic mobility
– Protein length
– Protein charge
Utility of SDS-PAGE
• Estimate relative molecular mass of proteins
• Determine relative abundance of proteins
• Determine the distribution of proteins among
fractions
• Assess progress of fractionation/purification
• Specialized techniques: Western blotting, twodimensional electrophoresis, peptide
mapping, etc.
• And much, much more…
SDS: Sodium Dodecyl Sulfate
• SDS: anionic detergent
– Linearizes proteins (breaks 2° and non-disulfide-linked
2° structures)
– Coats proteins with negative charges
• Since SDS generally imparts an even distribution
of charge per unit mass, proteins fractionate by
approximate size during electrophoresis
• Generally used with β-mercaptoethanol (β-ME or
2-ME) or DTT
– Reducing agents: break disulfide bonds between
amino acids
Which aa forms disulfide bonds?
Why do we like polyacrylamide gels?
•
•
•
•
•
•
Synthetic
Thermo-stable
Transparent
Strong
Generally chemically inert
Can be prepared with a wide range of average
pore sizes
SDS-PAGE Players
• Acrylamide: slow, spontaneous autopolymerization occurs when
dissolved in water
– Forms long single-chain polymers
– P.S. acrylamide is a neurotoxin that can be absorbed through the skin –
wear gloves!!
• Bisacrylamide: cross links polyacrylamide chains to one another
• Chemical buffer: stabilizes pH
– I.e. Tris, Bis-tris, imidazole
• Counterion: balances the intrinsic charge of the buffer ion and
affects the electric field strength during electrophoresis
– i.e. glycine, tricine
• Ammonium persulfate (APS): source of free radicals used as an
initiator for gel formation
• TEMED: stabilizes free radicals, improves polymerization
– Increasing free radicals decreases average polymer chain length
– Decreased average polymer length means stiff, cloudy, crappy gels
The Laemmli System
• Buffers : different pH and composition
– Generates a voltage gradient and a
discontinuous pH between the stacking and
resolving gel
Lining them up at the starting line:
• Stacking gel: ~4% acrylamide gel (pH 6.8)
– Poured on top of a ~10% acrylamide
resolving gel
– Large pore size
– Concentrates proteins (large ones can catch
up with the small ones) on top of the
resolving gel
And they’re off!
• Resolving gel: 10% acrylamide gel (pH 8.8)
– Small pore size
– Proteins separated according to relative
molecular size
Sample Preparation
• Laemmli sample buffer contains
– SDS
– β-ME
– Glycerol
– Bromophenol blue dye
• Protein samples are diluted 1:2 in Laemmli
sample buffer and boiled for 5min.
What does this do?
Assemble, Load and Run
• https://www.youtube.com/watch?v=bdBXwuuwSBo
• The gel electrode assembly is placed in the
electrophoresis chamber
• Running buffer is added
– Tris
– Glycine
– SDS
• Samples are loaded and proteins electrophoresed
at ~200V for about 45min
PolyAcrylamide Gel Electrophoresis
• What size band are you looking for? How big is
your protein? In kilodaltons? In kilo…what now?
• Daltons: named for the good John Dalton (17661844)
– Standard unit used for indicating mass on an atomic
or molecular scale
– Value = 1.660538921 x 10-27
• 1/12th mass of an unbound neutral atom of carbon-12 in its
nuclear and electronic ground state
• Approximately equal to the mass of one nucleon (1g/mol)
• Average mass of a single amino acid = 110 daltons
PolyAcrylamide Gel Electrophoresis
• For proteins 30-90kD using an 8-10% gel, the
bromophenol blue dye should travel to the
bottom of the resolving gel
• Perhaps you’re wondering what percent gel
we like to use for different sized proteins?
– 50-500kD:
– 20-300kD:
– 10-200kD:
– 3-100kD:
7% acrylamide
10% acrylamide
12% acrylamide
15% acrylamide
Disassembly and Staining
• Plates are separated and gel is dropped into a
staining dish containing deionized water (for a
quick rinse)
• Water is poured off and stain added
• Staining usually requires incubation overnight,
with agitation
– Agitation circulates the dye, facilitating
penetration and helps ensure uniformity of
staining
Staining SDS-PAGE Gels
• Coomassie Blue in methanol and acetic acid
– Acidified methanol precipitates the proteins
– Dye penetrates the entire gel but only sticks
permanently to the proteins
• Other staining methods: silver, zinc, fluorescent
dyes…
• Destaining: dye penetrates the entire gel but only
sticks permanently to the proteins
– Acetic acid/methanol with agitation:
• 50% methanol, 10% acetic acid: shrinks the gel, squeezing
out much of the liquid component
• 7% methanol, 10% acetic acid: gel swells and clears