1. Expression
Image Reference:

2. Induction

3. pLATE31 Vector pLATE Vector Landmarks T7 Promoter LacO RBS
His*Tag coding sequence
Multiple cloning sites
lacI coding sequence
bla(ApR) coding sequence

4. The Lac Operon

5. 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
Natural selection has favored bacteria that express
Only certain genes
Only at specific times when the products are needed by the cell
How does a bacteria selectively turn their genes on and off?
Lac operon: coordinates the expression of genes that produce enzymes used to break down lactose
The lactose operon is turned on by removing the repressor a sort of double negative.
Lac operon codes for three genes:
LacZ: codes for β-galactosidase (lactose  glucose and galactose)
Lactase is part of the β-galactosidase family of enzymes
LacY: codes for β-galactoside permease (pumps lactose into cell)
LacA: codes for β-galactoside transacetylase

6. 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?

7. 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

8. The Lac Operon
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.
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.
Part of the induction protocol states to not grow up cells overnight because genes will leak product (and the antibiotic will become depleted from the media). However, if you insist on growing overnight cultures, they suggest adding % glucose. Why?
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.

9. 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.

10. 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.

11. 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!!!

12. 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 (~ 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?

13. Protein Extraction

14. Where is my protein? In the medium? In the periplasm?
Target protein leakage from cells
Prolonged induction
Target protein is exported
In the periplasm?
Vector/target protein includes signal sequences
Image reference:
E coli has an outer and an inner membrane
What organelles in eukaryotes are made up of the same?
Periplasm: signal sequences
Signal recoginition particle (SRP) dependent
Signal sequences have high hydrophobicity and are involved in a rapid, cotranslational pathway
Non-SRP dependent
Promote export by a post-translational mechanism
Target protein: may contain a sequence recognized by SecB (major chaperone of export)
Cytoplasm: made up of water, salts and organic molecules
Soluble: polar proteins
Insoluble: nonpolar, hydrophobic proteins
In the cytoplasm?
Soluble
Insoluble

15. 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?

16. 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
Protease Inhibitors
Aprotinin, benzamidine, PMSF: inhibits serine proteases
Serine proteases: cleave peptide bonds in proteins, serine serves as the nucleophilic amino acid at the enzyme’s active site
EDTA: chelating agent, deprives proteases of their metal ion cofactors
Leupeptine: inhibits cysteine and serine proteases
Pepstatin A: inhibits aspartic proteases
Other additives
Glucose/sucrose: why is it a good idea to stabilize lysosymal membranes: reduces protease release

17. Protein Purification Protocol
After harvest, freeze the cells
Thaw cells and resuspend in lysis buffer
Tris-HCl + glycerol + NaCl
Add lysozyme
Incubate
Centrifuge
Tris-HCl: buffer
Glycerol: reduces non-specific interactions between proteins, protects proteins during freezing
NaCl: maintain the ionic strength of the medium
Why no SDS? Used freeze/thaw to disrupt cell membranes
Lysozyme: breaks down cell wall
Where is my protein?
Where are all the other (soluble cytoplasmic) cell proteins?

18. Tags and Affinity Chromatography

19. 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
Affinity chromatography
Ligand: An ion or molecule attached to a metal atom by coordinate bonding
Coordinate bonding: A covalent chemical bond between two atoms that is produced when one atom shares a pair of electrons with another atom lacking such a pair
Commonly used affinity columns:
Glutathione  GST (glutathione s-transferase)
Tripeptide binds GST which is generally fused to a gene of interest
Protein A/G: surface protein expressed Staphylococcal bacteria
Binds with high affinity to antibodies
Used for production of antibodies in bio-pharmaceuticals, most commonly bound to a stationary phase chromatography resin

20. Ni-NTA columns are a type of affinity chromatography
The high affinity of the Ni-NTA resins for 6xHis-tagged 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
Nickel has six electron coordination bonds. NTA binds to four of these bonds. Two ligand binding sites are then available to readily coordinate with His-tagged proteins
What does histidine look like? Where do you suppose the interaction takes place between Ni2+ and histidine?
How many protons does N have?
How many electrons does N need to be neutral?
How many electrons does N in histidine have after we pull off hydrogen?
Is it negatively charged or positively charged?

21. 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
Why is EDTA a bad idea for His-tag purification?
The high affinity of the Ni-NTA resins for 6xHis-tagged proteins or peptides is due to:

22. 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.
Protein elution
Why does reducing the pH help release histidine from Ni2+
What does reducing the pH do? (increses H+)
H+ pops back on nitrogen and is released

23. 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+

24. 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

25. SDS-PAGE

26. 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?
What determines the net charge of proteins? Amino acid composition
What determines the molecular radius of a protein? It’s shape
Shape is determined by secondary and tertiary interactions

27. SDS-PAGE Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
Developed by Ulrich K. Laemmli (Nature 227: , 1970)
Separate proteins according to their electrophoretic mobility
Protein length
Protein charge

28. 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, two-dimensional electrophoresis, peptide mapping, etc.
And much, much more…
Distribution of proteins among fractions
Cytoplasm: insoluble and soluble
Periplasm
Media

29. 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
Dodecyl: dōd-ə-ˌsil
SDS: anionic detergent (meaning when dissolved its molecules have a net negative charge within a wide pH range)
Which electrode do proteins migrate to in SDS-PAGE?
Reducing agent: what does that mean?
Draw cysteine (-CH2-SH)
What does it mean to reduce (OiL RiG)
Gain electrons (hydrogens)
Disulfide bonds: what kind of bonds? Between what amino acid?
Reducing agents: break disulfide bonds between amino acids
Which aa forms disulfide bonds?

30. 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

31. 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
Acrylamide and bisacrylamide: acrylamide forms single-chain polymers (viscous but does not form a gel)
Gel forms when bisacrylamide cross-links acrylamide polymers together

32. 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
Acrylamide concentration of the gel varies (generally 5%-25%):
Lower percentage gels better for resolving very high molecular weight proteins
Higher percentage gels are needed to resolve smaller proteins
Discontinuous buffer system: enhances the sharpness of the bands in the gel
Ion gradient is formed in early stages of electrophoresis: causes all proteins to focus into a single sharp band
Formation of ion gradient achieved by choosing a pH value at which the ions of the buffer are only moderately charged compared to the SDS-coated proteins (stacking gel)
Resolving gel has a pH value in which the buffer ions carry a greater charge (on average), causing them to ‘outrun’ the SDS-covered proteins and eliminate the ion gradient and stacking effect
Glycine and Cl-: can exist as positive, negative or neutral
pH 8.3: glycine negative, forced to enter stacking gel
pH 6.8: glycine mostly neutral, move very slowly
Cl- move more quickly, ahead of glycine, form an ion front
The two form a narrow zone with a steep voltage gradient
Proteins have an electrophoretic mobility between the two and concentrate in the narrow region
pH 8.8: glycine negative, outruns the protein, proteins are left in a very narrow band at the interface of the stacking and running gels
What would happen if we had no stacking gel?
Gel wells are ~1cm deep, need to fill them substantially to get enough protein onto the gel
Result in a smear from proteins entering the gel at different times

33. 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.
Glycerol: what does it do?
Increases the density so that when the sample is loaded it sinks to the bottom of the well
Bromophenol blue: monitor the electrophoresis process
For 30-90kD proteins using a 8-10% gel, bromophenol blue dye should travel to the bottom of the resolving gel
What does this do?

34. Assemble, Load and Run
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

35. 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 ( )
Standard unit used for indicating mass on an atomic or molecular scale
Value = 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
John Dalton: english chemist, meteorologist and physicist
Best known for his pioneering work in the development of modern atomic theory
Figure out how big the protein is:
How many nucleotides from start codon to stop? ( )
How many amino acids? (1365/3 = 455)
How many kD? (455*110 = daltons = 50kD)

36. 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: 7% acrylamide
20-300kD: 10% acrylamide
10-200kD: 12% acrylamide
3-100kD: 15% acrylamide

37. 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

38. 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