W9 Protein Expression and Purification

Question 1

minimal requirement for effective gene expression

Answer 1

presence of strong and regulatable promoter sequence upstream of the cloned gene

Question 2

problems of constitutive overexpression

Answer 2

creates energy drain detrimental to host cell

plasmid may be lost after several division cycles as cells without plasmid grow faster than those having one

sometimes proteins form insoluble aggregates > trap in inclusion bodies

Question 3

what is a protein fusion

Answer 3

many foreign proteins have low expression in heterologous host cells because of degradation > engineer dna construct such that foreign protein is in frame with a stable host protein > resultant is a fusion protein

Question 4

advantages and drawbacks of fusion protein

Answer 4

prevent degradation by host cell proteases

promote proper folding and protein solubility

facilitate expression of protein fragments

facilitate downstream protein purification

aid in detection or tracking

drawback: may mask protein of interest in some studies

Question 5

why is there a need to cleave the fusion protein

Answer 5

presence of host protein segment makes many fusion proteins unsuitable for clinical usage

host protein segment may affect the biological function of POI

Question 6

types of liquid chromatography

Answer 6

affinity: ligand interaction

gel filtration: size and shape

ion exchange: charge

hydrophobic: surface hydrophobicity

reverse phase: total hydrophobicity