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BMIS 310 > Protein Purification > Flashcards

Protein Purification Flashcards

1
Q

Describe the flow of genetic information to primary sequence

A

The genetic information is stored in DNA, then transcribed into mRNA, And finally translated into protein primary sequence.

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2
Q

What does the primary sequence of a protein determine

A

The primary sequence of a protein determines its ordered or disordered native 3-dimensional structure, hence its function

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3
Q

What are recombinant proteins

A

By manipulation of genetic codes, we can produce whatever amino acid sequence. We can use lab-cultured cells / organisms to produce large quantities of recombinant proteins

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4
Q

Applications of recombinant proteins (3)

A
  1. Truncated proteins (part of a large protein)
  2. Fusion protein (two or more proteins linked in one peptide chain)
  3. Tagged protein (protein fused with a short peptide for identification or purification)
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5
Q

What are the three key macromolecules for protein biosynthesis

A

1) mRNA
2) Ribosome complete with auxiliary factors such as the elongation factor and GTP as the energy source
3) aminoacyl-tRNA

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6
Q

how are peptide bonds formed and broken down

A

formed by condensation
Broken down by hydrolysis

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7
Q

What are the sources of proteins

A

Natural sources- tissues, seeds, eggs, cultured cells, microorganisms, media etc. Only naturally abundant proteins are available this way

Recombinant proteins- produced in cultured genetically modified organisms. Wild-type, mutant any engineered proteins can all be made available in this way, and in large amount

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8
Q

How are proteins extracted? How do you choose the extraction method

A

Need to break up the cell. There are many ways:
Osmotic shock
french press
sonification
Digestions
homogenization

The best way is the most convenient for your purpose

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9
Q

What are the practical considerations during extraction

A

Condition of the solution must be mild to keep the protein of interest in its native state:
pH
Temperature
Reducing agent
EDTA
Ionic strength
proteolysis
Glycerol or sucrose

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10
Q

Describe the soluble and insoluble part of extract after centrifugation

A

Soluble part is the supernatant/ crude extract.

Insoluble part is the pellet, which is cell debris, higher density, forms a pellet in the bottom

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11
Q

How can you measure the protein concentration in the supernatant/ crude extract

A

Bradford or Biuret method to monitor the efficiency of cell lysis

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12
Q

What are the effects of salt on protein solubility

A

In the optimal amount of salt, solubility is maximized

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13
Q

What is salting out and salting in

A

Salting in: solubility is very low in the absence of salt. Solubility increases when a small amount is present

Salting out: solubility decreases with excess concentration of salt or other solute

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14
Q

how can fractionation be achieved by salting out

A

Two proteins may salt out at different concentrations of salt. As one protein become insoluble, you can then centrifuge out the precipitate from the soluble portion.

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15
Q

What does amino acid composition determine

A

Determines charges on proteins

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16
Q

what is the isoelectric point

A

pH at which the protein has a net neutral charge

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17
Q

What do salts do to protein aggregation

A

Salts keep proteins separated from each other

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18
Q

When is solubility lowest

A

solubility is lowest around pi

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19
Q

Describe protein interactions at the following pH and pI
pH>pI
pH=pI
pH<pI

A

Proteins attract through hydrophobic interactions when pH=pI

Proteins repel at other

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20
Q

Describe the stationary and mobile phase of chromatography

A

Stationary: beads contained in the column
Mobile: aqueous solution supplied from the inlet, flows through the beads and filters the outlets

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21
Q

Describe a typical chromatography system

A

A typical chromatography
system also has a pump, an in-line UV absorption detector, as well as a computer for controlling the system and for displaying the chromatogram

22
Q

Describe the UV absorption of proteins

A

UV absorption at 280 nm
Mainly by aromatic residues
Real-time monitoring

23
Q

Describe Biuret & Bradford assays

A

time consuming
Fractions collected first, then assayed

24
Q

Describe activity assays

A

Can tells if it is the target protein

25
Q

What is Coomassie Blue G-250

A

Dyes free proteins red-brown
dyes protein-bound blue
Absorbance at 595 nm is measured for quantification of protein concentration (Bradford)

26
Q

What is a chromatogram

A
27
Q

What is an SDS-PAGE

A

Polyacrylamide gel electrophoresis
Smaller proteins move faster
Stain with Coomassie blue
Sodium dodecyl sulfate (SDS) denatures proteins by alkyl chain binding to hydrophobic core
Sulphate groups cover protein with negative charges
Large proteins are impeded more by the pores in gel matrix

28
Q

T/F it is rare that proteins other than the enzyme bind the substrate with similar affinity

A

True

29
Q

Describe affinity chromatography

A

Protein mixture is added to a column containing a polymer-bound ligand specific for the protein of interest

Unwanted proteins are washed through the column

Protein of interest is eluted by ligand solution

30
Q

High affinity of the Ni-NTA resins for 6xHistidyl-tagged proteins or peptides is due to what two things

A

High affinity of the Ni-NTA resins for 6xHistidyl-tagged proteins or peptides is due to:
1) Ni ions are anchored to agarose beads
2) two empty valence of Ni

31
Q

What is histagged protein

A

Have a stretch of 6 or more consecutive histidine residues that interact tightly with Ni ion that is bound to a NTA bead

Naturally occurring protein does not have this feature and does not bind Ni ion tightly

32
Q

What is the specificity of the Ni-affinity chromatography reaction? What purity does it create?

A

Extremely high, allows protein purification to a purity of 95% in a single step

33
Q

4 steps of Ni2+-affinity chromatography

A
  1. Prepare and equilibrate the column with a binding/ running buffer at pH~7 and 500mM NaCl
  2. Load crude sample. the protein with engineered polyhistidyl tag will be bound to the beads. Other proteins will not be bound.
  3. Wash away non-bound sample components with the running buffer (wash with same solution)
  4. Elute his-tagged protein with an elution buffer with 300 mM imidazole and 500 mM NaCl. Collect the effluent. (elute with different solution)
34
Q

Why do we use imidazole to liberate the bound protein

A

Imidazole ring is part of the histidine structure and a perfect competitor for binding the metal ion and liberating the bound protein

35
Q

How are TAG-proteins eluted

A

Either by a compeititve ligand or by cleavage

36
Q

Why is it called cation exchange chromatography

A

.

37
Q

5 steps of anion exchange chromatography

A
38
Q

Common steps in affinity and ion-exchange chromatography

A
  1. Prepare the column and equilibrate with a binding buffer
  2. Load sample in the binding buffer
  3. Wash the column with the binding buffer
  4. Elute the bound protein with the elution buffer or a series of elution buffer with a concentration gradient
39
Q

Describe gel filtration chromatography (aka size exclusion, molecular sieving)

A

Larger molecules travels less into the pores and therefore elutes earlier
There are porous beads in the column, and small proteins enter and are slowed down

40
Q

Gel filtration procedure

A
  1. Prepare a column by filling with beads and flooding with a running buffer
  2. Load the protein mixture on the top
  3. Keep supplying the running buffer on the top

BIGGER ELUTES FASTER

41
Q

What is a gel filtration chromatogram

A
42
Q

Why do larger molecules migrat faster in gel filtration

A

The beads for gel filtration are made of porous
material
Detour effect:
- Huge molecules travel through gaps between effects
- Medium molecule detour into large enough cavities
- Tiny molecules detour into all cavities

43
Q

What type of column will result in better separation for GF

A

Bigger the column will result in better separation. The wider the molecules can spread

44
Q

Why can we only estimate relative molecular weights from chromatography

A

shape affects the pore-entering possibility of a molecule, elongated molecules are less likely to enter pores

If proteins have a known round shape, then MW = RMW

45
Q

What the partition coefficient (Kav)? When do bigger molecules, smaller molecules, and intermediate molecules elute?

A

Independent of column size as long as we use the same type of beads

(Ve- Vo)/(Vt-Vo)

46
Q

MW range for G-200, G-100, G-25

A

– G-200 good for 5,000-600,000 Daltons
– G-100 good for 4,000-150,000 Daltons
– G-25 good for 1,000-5,000 Daltons

47
Q

How to choose running buffer

A

Retains solubility of the sample
Has 100 mM or more salt to suppress charge-charge interaction between proteins
Column has to be equilibrated by the running buffer by at least Vt

48
Q

Which chromatography should be used first

A

most specific (powerful) chromatography should be applied first

1st: Affinity
2nd: Ion Exchange
Last: Gel filtration

49
Q

How can we concentrate protein solutions

A

Protein solution can be concentrated by ultra-filtration. A filter with a cutoff size (5000 Dalton for example) is used. The protein solution is passed through this very slow filter in a nitrogen pressure cell or a centrifugation tube

50
Q

Describe the storage of proteins

A

Protein solution can be stored at -80C for an extended period of time. At first, 10% glycerol is usually added to the protein solution as the cryoprotectant. Then the sample is transferred into many small sealed tubes, and the tubes are dropped into liquid nitrogen for rapid cooling. Finally, the cooled sample tubes are stored in a -80C freezer.
There are other ways of storing proteins. The above-mentioned one is the most popular.

51
Q
A

BMIS 310 (8 decks)

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