Recombinant DNA Basics #2

Question 1

How are proteins purified?

Answer 1

The transcribed sequence contains a polyhistidine tag which will bind strongly to Ni2+ immobilized on crosslinked agarose beads

nonbinding proteins are washed away. Tagged protein is then washed with high concentrations of imidazole which is removed by gel filtration

HisTag can be removed using a protease

Question 2

How is recombinant DNA introduced into cells?

Answer 2

electroporation
heat shock
conjugation
lipofection
calcium phosphate coprecipitation
microinjection
viral infection

Question 3

How do different host organisms for protein production compare?

Answer 3

Prokaryotic bacteria - high concentration, low MW, but is limited by S-S bridges, misfolding, and no glycosylation

Eukaryotic yeast - high concentration, high MW, and glycosylation is possible

Eukaryotic mammalian cells - low concentration, high MW, unique glycosylation patterns can have application

Question 4

What are the pros and cons of using E. coli as a host for protein production?

Answer 4

Adv - fast growing, simple nutrient mix, affinity purification, better protein solubility, and better folding/protein secretion

Disadv - lack of eukaryotic post translational modifications, surface antigens, accumulation of inclusion bodies if folding problems arise

Question 5

What are pros and cons of using yeast as an expression host?

Answer 5

adv - established genetics/fermentation, sufficient protein production, fast growth, post-translational modifications

disadv - hyperglycosylation, secreted yields are initially very low

Question 6

What are pros and cons of using mammalian cell lines as expression hosts?

Answer 6

adv - correct post-translational modification

disadv - complex, slow growth, typically low production, cell lines not robust, cell lines can be stolen without credit

Question 7

Why is protein glycosylation important in proteins?

Answer 7

if contributes to correct folding. Chaperones will find terminal Glc and will help contribute to the protein’s correct folding

Question 8

Define a GMO

Answer 8

organism whose genetic material has been altered using genetic engineering techniques.
can be transgenic - containing 1 or more genes from different species or cisgenic contains modified genes from the same species

Question 9

Describe the benefit of transgenic milk

Answer 9

potential production media

• established infrastructure for high volume production
• well-characterized media (milk) simplifies processing
• efficient targeted heterologous expression protein
• ongoing supply of product guaranteed through breeding