E. Coli

Question 1

Challenges when using E. Coli in recombinant protein expression

Answer 1

Folding mechanisms, post-translational mechanisms, not keeping target properties of the protein, solubility issues.

Question 2

Why are large proteins a problem for E. Coli recombinance?

Answer 2

They are multidomained (have 2 parts) e coli lacks the chaperone machinery to fold these.

Question 3

Why are hydrophobic and transmembrane proteins hard for e. coli to produce?

Answer 3

Problems with folding, leading to the formation of inclusion bodies.

Question 4

Inclusion bodies

Answer 4

Dense aggregates of misfolded proteins and can cause major implications.

Question 5

PTMs of human proteins

Answer 5

Glycosylation, phosphorylation and disulfide bond formation.

Question 6

Translational stalling

Answer 6

When E. coli stops producing a protein due to a human codon present in the sequence which E. coli is incapable of creating.

Question 7

What does E. coli do with misfolded proteins and inclusion bodies?

Answer 7

Either refolded using chemicals in vitro, or protease used by E. coli and nutrients re-used.

Question 8

Strategies to overcome E. coli limitations to produce human recombinant proteins.

Answer 8

Optimizing expression vectors, select an engineered strain, optimize conditions, remove unstable domains, in extreme cases solubilization of inclusion bodies can be done.

Question 9

Optimizing expression vectors

Answer 9

1.Only use strong promoters, such as T7 from pET system
2. low concentrations of IPTG can slow proteinsynthesis.
3. Optimize the sequence for E. coli, by using pRARE plasmids.
4. Adding fusion tags such as MBP (Maltose binding protein) or GST (glutathione S transferase) to improve folding

Question 10

pRARE

Answer 10

A plasmid designed to allow E.coli to express human proteins as it allows coding of rare tRNA codons that E. coli does not usually code. (plasmid Rare tRNA Expression)

Question 11

IPTG

Answer 11

Compound that slows proteinsynthesis by placing an overwhelming demand on the cells resources. (Isopropyl-b-D-1-thiogalactopyranoside)

Question 12

Strains of E.coli

Answer 12

1. BL21(DE3) standard strain, high expression
2. Rosetta(DE3) used where rare tRNAs are needed
3. Origami(DE3) used where complex folds and high amounts of disulfide bridges are present

Question 13

Optimizing growth conditions

Answer 13

1. lowering temps slows proteinsynthesis
2. rich media enhances growth and protein yield
3. Supplementation with arginine and sorbitol to reduce aggregation.

Question 14

Refolding and Purification from inclusion bodies

Answer 14

1. Chaotropic agents such as urea and guanidine hydrochloride dissolve mishapen proteins, amino acids re-used.
2. Use a buffer like redox paired glutathione to promote proper folding.

Question 15

Protein Engineering

Answer 15

Remove truncation regions in sequence, and introduce point mutations to reduce hydrophobic patches or stabilize proteins.