Protein expression and purification

Question 1

why express and purify recombinant proteins

Answer 1

ethics of purifying from human tissues

genetically engineer microbes i.e. E coli
to produces
recombinant proteins

experiment probing structure/function require the protein of interest to be pure

probing activators/inhibitors on PoI’s function

study structure via cryo-EM, Xray crystallography or NMR

Question 2

directional cloning pET-3a typical expression vector

Answer 2

NdeI upstream
BamHI downstream
stop codon TAA

cut plasmid with restriction endonuclease it would linearised plasmid and get the amplified PCR product ligated into cloning vector = join sticky ends

recombinant vector transformed into competent + grow

Question 3

how to express a recombinant protein from an expression vector

Answer 3

T7 promoter = T7 RNA polymerase makes mRNA in bacteria

we dont want expression of the protein during cloning as we don’t yet know how it works and if it could have a negative result

host cell for cloning without T7 RNA polymerase

BL21 cells = host cells for expression expresses T7 DNA polymerase

Question 4

importance of regulation of expression

Answer 4

foreign gene products may be toxic to the host so constitutive expression of a gene in a foreign host can cause cell death or plasmid DNA rejection

can control:
rate at which gene is transcribed by the polymerase into the mRNA

Question 5

analysing protein expression by SDS page

Answer 5

shows recombinant protein expression after induction with IPTG
the recombinant protein is soluble in the cell

samples are denatured using SDS which unwinds the tertiary structure back to the primary structure

electrophoresesed them on a poly acrylamide gel stained with coomassie blue

separates by size and abundance of proteins in your sample

sample of each stage run next to each other

Question 6

protein purificatin

Answer 6

for a sample of a protein of interest purification involves the removal of contaminants that may interfere with analysis of that protein of interest
activators increase POT acitiviy inhibitors proteases unfolded/degraded molecules of POI

how:
homogenisation and cell lysis disperese the tissue and break open cells using lysozyme followed by crude filtration

spin the homogonate in a centrifuge to fractionate and select the right subcellular component

then purify depending on chemical physical or biological properties
sequentially purify the protein
i.e.
1. solubility in salt = salt precipitation
2. size- gel filtration or size exclusion chromatography
3. charge - ion exchange chromatography
net positive or net negative and then stick to a charged surface

4. hydrophobic nature - reverse phase high pressure liquid chromatography
5. bio-specificity
   affinity chromatography using a tag

with each purification step the purity should increase but loss of protein is inevitable due to proteolytic activity during cell lysis or protein may be unstable at high temperatures so can be kept in ice or instability of pH, chemical interfaces between glass and plastic can denature protein and divalent metal ions covalently binding to thiol groups= denaturation)

Question 7

determining POI

Answer 7

we need assays to determine how many units of the POI there are and what the total conc of protein there is

test using knowing characteristics i.e. molecular weight using SDS-page

yield = amount of POI after each purification step
required yield depend on how much protein is needed

detection using antibodies against POI with tag - nanogram
protein sequencing/identification by mass pec sub microgram
structural studies
x ray crystallography - tens of milligrams

minimise no. of handling steps = maximise yield
3-5 chromotogrpahy steps

Question 8

assaying

Answer 8

PURE=
the protein conc. can be calculates using absorbance spectroscopy from the absorbance at w 280 if the molar extinction coefficient is known

if A280 is 1 protein conc = 1mg/mL roughly

IMPURE
biuret assay
Lawry assay
bicinchonic acid BCA Assay - precipitate protein = colour change
Bradford coomassie assay - looking for the total conc of protein in each step

Question 9

measuring protein conc w the Bradford coomassie assay - looking for the total conc of protein in each step

Answer 9

uses a coomassie blue dye which binds very strongly to hydrophobic amino acids like arginine by burying self in the protein surfacepermenantly

polycyclic aromatic

absorbs at 595nm when bound to protein

results can be used to construct a standard curve using a cheap protein Bovine Serum Albumin
one can take unknown sample conc perhaps half way through exp and want to knowhow much conc you have..
by using curve to make estimation because BSA is a good average protein, so you take its absorbance reading at wavelength 595 and read down to the concentration on the x axis in micro grams per 10microlitres

Question 10

quantify protein purification

hoping for increasing activity and increasing fold purity as yield goes down

Answer 10

tubulate:

step no.

procedure name

volume ml

[protein] (mg/ml) from Bradford Assay

Activity = (U/ml) from enzyme assay

total protein (mg) =
[protein] (mg/ml) x Volume (ml)

Total activity (U) = Activity (U/ml)) X vol (ml)

specific activity per mg of protein (U/mg) = Activity (U/ml) / [protein] (mg)

fold purity
relative to step 1

= specific activity at step n over specific activity at step 1

2 times more pure than step 1

% yield
total activity at step n over Total activity at step 1 x 100

1U = 1micro mol product formed per min

Question 11

protein properties that enable purification

Answer 11

1. differential solubility =
   ammonium sulphate salt precipitation

2 molecular mass = gel filtration or size-excision chromatography

3. surface charge
   ion exchange chromatography or isoelectric focussing
4. hydrophobic nature
   reverse-phase high pressure liquid chromatography or hydrophobic interaction chromatography
5. biospecificity = affinity chromatogrpahy