Biochemistry Lab Protocol Questions: 1

Front 1

Inclusion Body

Back 1

Inclusion bodies are densely packed, insoluble aggregates of unfolded protein. It is common for overexpressed proteins to form inclusion bodies.

Front 2

Why might we want our protein to be in the form of inclusion bodies?

Back 2

In the case of RNase A, inclusion bodies are desirable so as to prevent unwanted degradation of RNA

Front 3

Why wouldn't we want inclusion bodies?

Back 3

For many proteins (not RNase A), it is extremely difficult to recover native (active) protein from inclusion bodies.

Front 4

What does the inclusion body denaturing solution do?

Back 4

7 M guanidinium-chloride, 50 mM Tris-HCl pH 7.5, 10 mM DTT

Front 5

Why do we add acetic acid to denatured inclusion bodies?

Back 5

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.

Front 6

Why do we dialyze protein solution the first day against 20 mM acetic acid ON @ 4ºC?

Back 6

Since we denatured our protein w/ salt (guanidinium chloride), placing dialysis bag in acetic acid should remove more impurities, especially the salt.

Front 7

Refolding Buffer

Back 7

(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

Front 8

Equilibration buffer

Back 8

50 mM NaOAc, pH 5.2, 10 mM EDTA

Front 9

Column wash buffer

Back 9

50 mM NaOAc, pH 5.2, 10 mM EDTA, 100 mM NaCl

Front 10

Elution buffer

Back 10

50 mM NaOAc, pH 5.2, 10 mM EDTA, 300 mM NaCl

Front 11

What type of protein purification did we do to our dialysate?

Back 11

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.

Front 12

Equilibration buffer

Back 12

50 mM NaOAc, pH 5.2, 10 mM EDTA

* Pumped through column to quilibrate the opposing charged ions

Front 13

Other Buffers:

Column wash buffer:100 mM NaCl
Elution buffer: 300 mM NaCl
Column cleaning buffer:1M NaCl

Back 13

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

Front 14

After removal of dialysis bag from refolding buffer, we add 6 drops of glacial acetic acid to your protein solution. Why?

Back 14

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.

Front 15

How do you test if a pipet is properly calibrated?

Back 15

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

Front 16

What is an endonuclease?

Back 16

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

Front 17

Why do we use restriction enzymes (endonucleases)?

Back 17

Used to cleave DNA molecules into specific fragments that are more readily analyzed and manipulated than the entire parent molecule.

Front 18

Contents of Polymerase Chain Reaction
(PCR)

Back 18

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

Front 19

Gel Electrophoresis

Back 19

* 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.

Front 20

What is RNase A?

Back 20

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

Front 21

What is Angiogenin?

Back 21

Promotes blood vessel growth. Acts in nucleus. Thought that it is regulated by RI (in cytoplasm) if it "leaks" out.

Front 22

Ribonuclease Inhibitor (RI)

Back 22

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.

Front 23

pKa of RI

Back 23

4.7

Front 24

Gel Electrophoresis Setup:

Back 24

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

Front 25

Goal of Restriction Digest Experiment

Back 25

To analyze a sample of plasmid DNA (pET-19b) by gel electrophoresis

Front 26

BamH 1 (319)

Back 26

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.

Front 27

Nde1 (331)

Back 27

Nde1 = Restriction enzyme
331 = base that Nde1 cuts

Front 28

Black large arrow

Back 28

Represents area with multiple cloning site (MCS)

Front 29

Bold Arrow for pET-19b

Back 29

unique to this plasmid, therefore a good place to cut and/or insert

Front 30

Regular arrow on plasmid map

Back 30

direction of transcription

Front 31

How does uncut DNA run?

Back 31

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

Front 32

What does doing a single BamH1 cleavage do to our DNA running on the gel?

Back 32

The single BamH1 cleavage makes the plasmid linear, giving us a better estimate of its size ("true coefficient")

Front 33

Why do we add 20mM acetic acid slowly to our denatured protein solution?

Back 33

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.

Front 34

How to create 1° stock solution:

Back 34

We want a concentration of 1ng/ul:
we have 200ug:
200ug/200ug = 1000ng/ul
Therefore, add 200uL of water to primer

Front 35

How to create 2° stock solution:

Back 35

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

Front 36

What goes into the PCR thermal cycler?

Back 36

* Rxn buffer
* plasmid
* primer 1
* primer 2 (complementary)
* dNTP
* water

Front 37

Where does RNase A bind?

Back 37

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

Front 38

What types of interactions bind RI to RNase A?

Back 38

Mostly electrostatic interactions. Also some H-bonds. and salt bridges (ionic).

Front 39

Are inclusion bodies in RNase A desirable?

Back 39

YES
Because it prevents unwanted degradation of RNA.

Front 40

dNTP

Back 40

solution of bases (AGCT)

Front 41

How many PCR cycles does a single amino acid change require?

Back 41

16

Front 42

What is the buried surface of the RNase A - RI complex composed of?

Back 42

49% non-polar
27% polar
24% charged
(this is slightly more charged than usual)

Front 43

How many H-bonds and salt links connect RI and RNase A?

Back 43

18 (11 involve at least one charged partner)

Front 44

How does RI inhibit RNase A activity?

Back 44

Prevents the access of substrates to the active site by steric hindrance; does not mimic RNase-RNA interaction
(i.e. indirect antagonist)

Front 45

Why might RI inhibit RNase A?

Back 45

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.

Front 46

What happens to RI when bound to RNase A?

Back 46

It exhibits the flexibility of its structure by conformationally adapting to the RNase A.

Front 47

Thermal Cycler of PCR

Back 47

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.