Protein purification

Question 1

Requirements for protein purification

Answer 1

Fast with few steps

Inexpensive

High yield

PURE protein

Question 2

Facts good to know before protein purification

Answer 2

Molecular weight

Trp content

Extinction coefficient

Stability

Known protein structure

Isoelectric points (PI)

Known ligands / Inhibitors

Question 3

Sulfonamides

Answer 3

Competitive inhibitor for carbonic anhydrase

Question 4

Fractionated centrifugation

Answer 4

After the initial tissue maceration the solution is filtered to remove large debris and centrifuged at low speed to remove unbroken cells (<1000g).

PROGRESSIVE increase in centrifugal force pellet sampler cellular organelles and eventually at highest speeds. Large protein Complexes may be isolated.

Question 5

Methods of purification

Answer 5

Fractionated precipitation

Ion-exchange chromatography

Gel-filtration

HIC (Hydrophobic interaction chromatography)

His-tag chromatography

Question 6

Salting in

Answer 6

Refers to the effect where increasing the ionic strength of a solution increase the solubility of a solute.

Question 7

Salting out

Answer 7

Is when at high concentration of salt, the solubility of the proteins drop sharply and proteins can precipitate out.

Question 8

Hofmeister series (Cation)

Answer 8

Stabilizing ions

Kosmotropic

Increase surface tension

Decease solubility of non-polar side-chain

salting out

Question 9

Hofmeister series (Anions)

Answer 9

Destabilizing ions

Chaotropic

Non effects on surface tension

Increase solubility of non-polar side-chain

salting in

Question 10

Isoelectric point

Answer 10

PH < PI positive

PH = PI Zwitterion

PH > PI negative

Question 11

Cation exchange

Answer 11

Increase ⟦Salt⟧ and PH will free the protein

Question 12

Anion exchange

Answer 12

Increase ⟦Salt⟧ and Decrease PH will free the protein

Question 13

Gel Filteration

Answer 13

Large protein comes out frist

Question 14

HIC

Answer 14

Separates proteins on the relatives hydrophobicity

Column matrix has hydrophobic groups covalently attached

Proteins binds to an HIC column under high salt concentrations and eluted in low salt concentration or with PEG

Question 15

pET-28a (+) expression system

Answer 15

DE3 lysogen encodes T7 RNA polymerase

Lacl Q gene is always expressed and protein product binds both operators when IPTG is absent

cloned gene is only transcribed by T7 RNA polymerase enzyme in presence of IPTG

Absence of IPTG
*lacl protein binds to both operator sites (T7 and your cloned gene) both gene are silent.

IPTG added

• IPTG binds to lacl and Lacl protein leaves both operator sites
• T7 polymerase gene is transcribed / translated
• T7 polymerase enzyme binds to T7 promoter site and your gene is expressed

Question 16

Detection methods to analyze the purity of sample

Answer 16

SDS PAGE

Western blot: primary antibody binds to target and enzyme conjugated secondary antibody binds to the primary.
*Detection signal are colorimetric or chemiluminescent

Mass spectrometry