Bacterial protein production systems

Question 1

Is e.coli gram negative? Do gram-negative bacteria secrete proteins?

Answer 1

Yes, No.

Question 2

Do gram-negative bacteria have cell walls? Is e.coli gram-negative?

Answer 2

Yes, Yes.

Question 3

List advantages of using e.coli for protein purification.

Answer 3

1. Genetics and biochemistry is understood.
2. Easy to manipulate.
3. Grows fast.

All the above lead to a large yield.

Question 4

List disadvantages of using e.coli for protein purification.

Answer 4

1. No intron-splicing mechanism.
2. No post-translational modifications.
3. Endotoxin (LPS) - BIGGEST DOWNSIDE.
4. Not generally regarded as safe (GRAS).

Question 5

What bacteria can exchange e.coli if protein is thought to be interacting with LPS? Any downsides?

Answer 5

Lactococcus lactis. Gram+.

Positive: Secretes proteins

Downsides: Lots of proteases.

Question 6

List different antibiotic resistance genes.

Answer 6

1. bla –> beta-lactamase –> Ampicillin resistance.
2. tet –> membrane transporter –> tetracycline resistance.
3. kan –> aminoglycoside acetyltransferase –> kanamycin resistance.

Question 7

What does “host range” mean (plasmids)

Answer 7

The range of subspecies in which the plasmid is stable.

Question 8

Name RNA polymerase (RNAP) subunits.

Answer 8

1. Alfa
2. Beta - catalytic center
3. Sigma
4. Omega

If all comes together, it’s called a holoenzyme.

Question 9

There are two trasncription stoppages in e.coli, which ones?

Answer 9

Rho-dependent or -independent.

Question 10

Is the shine-dalgarno sequence relevant in replication, transcription or translation?

Answer 10

Translation. Shine-dalgarno sequence = ribosomal binding site.

Question 11

What’s the importance of N-formyl-methionine?

Answer 11

When translating a protein, the first aa-residue is N-formyl-methionine. There are two enzymes which either modulate the residue or cleave it away. This is important when chosing bacterial strain.

Question 12

Insert question about Lac-system if you have time when going through these.

Question 13

How is the T7 polymerase system regulated?

Answer 13

(0.) You can tighten the regulation by adding aT7 lyzosyme plasmid. These generate T7 lysozyme. When transcribed, the lysozyme degrades T7 polymerase.

1. LacI is constitutively produced within the cell. LacI inhibits the genomic promoter which promotes T7 pol (in the DE3 region).
2. When IPTG is present, LacI dissociates from the promoter. This leads to T7pol being transcribed.
3. The T7pol transcribes any genes which has its promoter; this could be a plasmid.If it’s a plasmid, it should include the gene for LacI which in turn inhibits the T7pol after it has performed its function.

If there’s a T7 lysozyme plasmid transformed, the T7 lysozyme will be produced, which degrades the T7 polymerase.

Question 14

Make a question about the pBad expression system if you have time when doing these.

Question 15

Why is it hard getting rid of potein degradation in the cells when trying to purify protein?

Answer 15

Proteases are integral for cell health, when downregulating them, the ells may die.

You can force inclusion bodies, fuse another protein to your peotein of interest and optimise pH. All of the aforementioned methods reauire lots of emperic data and engineering

Question 16

Name ways to control proteolysis.

Answer 16

1. Steric hindrance via fusion proteins.
2. Forced inclusion bodies.
3. Co-expression of protease-inhibitor.
4. pH-optimisation.