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47 Cards in this Set

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Inclusion Body
Inclusion bodies are densely packed, insoluble aggregates of unfolded protein. It is common for overexpressed proteins to form inclusion bodies.
Why might we want our protein to be in the form of inclusion bodies?
In the case of RNase A, inclusion bodies are desirable so as to prevent unwanted degradation of RNA
Why wouldn't we want inclusion bodies?
For many proteins (not RNase A), it is extremely difficult to recover native (active) protein from inclusion bodies.
What does the inclusion body denaturing solution do?
7 M guanidinium-chloride, 50 mM Tris-HCl pH 7.5, 10 mM DTT
Why do we add acetic acid to denatured inclusion bodies?
The acetic acid causes a gradual change in pH, causing impurities to precipitate from the protein solution. RNase A is stable in this acidic environment.
Why do we dialyze protein solution the first day against 20 mM acetic acid ON @ 4ºC?
Since we denatured our protein w/ salt (guanidinium chloride), placing dialysis bag in acetic acid should remove more impurities, especially the salt.
Refolding Buffer
(100 mM Tris-HCl pH 8.0, 100 mM NaCl, 10 mM EDTA, 1.0 mM reduced glutathione, 0.2 mM oxidized glutathione

We place our protein solution in refolding buffer after it has been dialyzed against acetic acid, spun, and then supernatant collected to allow the protein to fold into its correct conformation
Equilibration buffer
50 mM NaOAc, pH 5.2, 10 mM EDTA
Column wash buffer
50 mM NaOAc, pH 5.2, 10 mM EDTA, 100 mM NaCl
Elution buffer
50 mM NaOAc, pH 5.2, 10 mM EDTA, 300 mM NaCl
What type of protein purification did we do to our dialysate?
Ion exchange chromatography

* Anion exchange resins have a positive charge and are used to retain and separate negatively charged compounds

* We added successively larger molarities of NaCl (100mM, 300mM, 1M).

*The most weakly charged compounds will elute first, followed by those with successively stronger charges.
Equilibration buffer
50 mM NaOAc, pH 5.2, 10 mM EDTA

* Pumped through column to quilibrate the opposing charged ions
Other Buffers:

Column wash buffer:100 mM NaCl
Elution buffer: 300 mM NaCl
Column cleaning buffer:1M NaCl
1. Column Wash Buffer
* [lowest] NaCl: 100mM
* elutes barely charged compounds
2. Elution buffer
* [moderate] NaCl: 300mM
* eludes moderately charged compounds
3. Column cleaning buffer: 1M
* eludes highly charged compounds
After removal of dialysis bag from refolding buffer, we add 6 drops of glacial acetic acid to your protein solution. Why?
Adding this acid will decrease the pH to 5.2, causing the deprotonated cysteine S: to be protonated into S-H, so protein will not reform disulfide bonds.
How do you test if a pipet is properly calibrated?
1. Tare a microfuge tube on an analytical balance.

2. Pipet water into the tube to determine if it is functioning correctly.

** 1ml should weight 1 gram
What is an endonuclease?
Restriction Enzyme:
* cleave double-stranded DNA at specific sequences
* biological role = cleave foreign DNA molecules
* recognize specific sequences (4-8bp long) : they hydrolyze a phosphodiester bond in eah strand in this region
Why do we use restriction enzymes (endonucleases)?
Used to cleave DNA molecules into specific fragments that are more readily analyzed and manipulated than the entire parent molecule.
Contents of Polymerase Chain Reaction
(PCR)
1. DNA template = contains region of DNA fragment to be amplified
2. Two primers = determine beginning and end of the region to be amplified
3. Polymerase = copies the region to be amplified
4. Bases = from which the DNA Polymerase builds the new DNA
5. Buffer = provides chemical environment for the DNA Polymerase
Gel Electrophoresis
* Restriction fragments can be separated and displayed.
- mobility of DNA fragment is inversly prop. to log of #of base pairs.
- gel stained w/ Ethidium Bromide, which fluoresces orange when bound to double-helical DNA molecule.
What is RNase A?
Bovine Pancreatic Ribonuclease
- An endonuclease (restriction enzyme) that cleaves single stranded RNA.
- First model protein: showed that proteins can spontan. fold (based on 1⁰ structure)

* RNase A and its homologs have cytotoxic and cytostatic effects, particularly on cancer cells.

* The RNA substrate lies in this cleft and is cleaved by two catalytic histidines via a cyclic phosphate intermediate that is stabilized by nearby lysines
What is Angiogenin?
Promotes blood vessel growth. Acts in nucleus. Thought that it is regulated by RI (in cytoplasm) if it "leaks" out.
Ribonuclease Inhibitor (RI)
Large acidic (pI ~4.7), leucine-rich repeat protein that forms extremely tight complexes with certain ribonucleases.

* consisting of alternating α-helices and β-strands along its backbone -- horseshoe.

The parallel β-strands and α-helices form the inner and outer wall of the horseshoe, respectively.

Stabilized by buried Asp at the base of each turn, as it passes from α-helix to β-strand.
pKa of RI
4.7
Gel Electrophoresis Setup:
1. Place gel in rack (RUN TO RED +)
2. Cover w/ buffer (so that a thin layer covers gel)
3. Standard in center lane, then 1,2,3
4. Cover, Run @120 volts for ~40min -- until yellow band is almost to the end
Goal of Restriction Digest Experiment
To analyze a sample of plasmid DNA (pET-19b) by gel electrophoresis
BamH 1 (319)
BamH = Restriction enzyme
319 = base that it cuts

The enzyme makes two incisions, one through each of the phosphate backbones of the double helix without damaging the bases.
Nde1 (331)
Nde1 = Restriction enzyme
331 = base that Nde1 cuts
Black large arrow
Represents area with multiple cloning site (MCS)
Bold Arrow for pET-19b
unique to this plasmid, therefore a good place to cut and/or insert
Regular arrow on plasmid map
direction of transcription
How does uncut DNA run?
It is supercoiled - wound upon itself (very small), therefore it gives the illusion that it is smaller than reality on the gel b/c it has a smaller frictional coefficient
What does doing a single BamH1 cleavage do to our DNA running on the gel?
The single BamH1 cleavage makes the plasmid linear, giving us a better estimate of its size ("true coefficient")
Why do we add 20mM acetic acid slowly to our denatured protein solution?
We add acetic acid to precipitate impurities. If we add the acid too fast, the rapid change in pH will precipitate the inclusion body, not just impurities.
How to create 1° stock solution:
We want a concentration of 1ng/ul:
we have 200ug:
200ug/200ug = 1000ng/ul
Therefore, add 200uL of water to primer
How to create 2° stock solution:
We want concentration of 25ng/uL
C₁V₁ = C₂V₂
1000ng/uL * V₁ = 25ng/uL * 200uL
V₁ = 5uL
Therefore, add 5uL 1° stock to 195uL molecular grade H₂O
What goes into the PCR thermal cycler?
* Rxn buffer
* plasmid
* primer 1
* primer 2 (complementary)
* dNTP
* water
Where does RNase A bind?
RNase A binds to the concave region of the inhibitor protein comprising its parallel ß-sheet and loops.

The inhibitor covers the ribonuclease active site and directly contacts several active-site residues
What types of interactions bind RI to RNase A?
Mostly electrostatic interactions. Also some H-bonds. and salt bridges (ionic).
Are inclusion bodies in RNase A desirable?
YES
Because it prevents unwanted degradation of RNA.
dNTP
solution of bases (AGCT)
How many PCR cycles does a single amino acid change require?
16
What is the buried surface of the RNase A - RI complex composed of?
49% non-polar
27% polar
24% charged
(this is slightly more charged than usual)
How many H-bonds and salt links connect RI and RNase A?
18 (11 involve at least one charged partner)
How does RI inhibit RNase A activity?
Prevents the access of substrates to the active site by steric hindrance; does not mimic RNase-RNA interaction
(i.e. indirect antagonist)
Why might RI inhibit RNase A?
ANase A is a secreted enzyme that should not normally come into contact with cytoplasmic inhibitor; RI may have a role in inhibiting traces of secretory RNases if they illegitimately foremd int he cytoplasm or leaked into the cytoplasmic compartment.
What happens to RI when bound to RNase A?
It exhibits the flexibility of its structure by conformationally adapting to the RNase A.
Thermal Cycler of PCR
PCR done in thermal cycler:

1.96°C - Denaturing = Separate the strands by breaking H-bonds b/w bases. Make sure everything is single-stranded.

2. Tm-5°(~55°C) - Annealing = Primers can attach themselves to the single DNA strands.

3. 72° - Elongation = DNA polymerase copies DNA strands. It starts at the annealed primer and works its way along the DNA strand.