Expression of recombinant proteins in E.coli

Question 1

Why do we express recombinant proteins in E.coli?

Answer 1

When E.coli divides, they will produce the produce the protein we want. Then we purify it out.

Question 2

What is a recombinant gene often linked to so that it can be expressed in E.coli?

Answer 2

Linked to a PROMOTER that E.coli recognises.

Linked to a RIBOSOME BINDING SITE that E.coli recognises.

Question 3

E.coli is not the only system we use to express recombinant proteins. What are some other ways?

Answer 3

Plant cells, mammalian cells, animal cells and yeast.

Question 4

Name some protein based therapeutics.

Answer 4

Hormones, cytokines, vaccines and monoclonal antibodies.

Question 5

The Lac Operon comprises of 3 structural genes. What are they?

Answer 5

LacZ- codes for B-galactosidase
LacY- codes for lactose permease
LacA- codes for transacetylase

Question 6

The Lac Operon comprises of 3 regulatory genes. What are they?

Answer 6

LacP- promoter sequence
LacI- codes for the lac repressor gene
LacO- operator sequence

Question 7

What is the function of B-galactosidase?

Answer 7

Hydrolyses the glycosidic bond between glucose and galactose (breaks down lactose)

Question 8

What is the function of lactose permease?

Answer 8

Makes the cell permeable to lactose

Question 9

What is the cis-acting sequence of the lac operon?

Answer 9

consists of LacP, LacO and the ribosome binding site

Question 10

What is the trans-acting sequence of the lac operon?

Answer 10

LacI

A gene that produces something that has an effect away from the gene

Question 11

What is the function of the lac-repressor?

Answer 11

Regulates the lac operon by stopping transcription by blocking DNA pol from binding
It is inactive in presence of lactose
It is active in absence of lactose

Question 12

What is an operon?

Answer 12

A sequence of genes working together for the same function/purpose

Question 13

What is the most popular system used to express recombinant proteins in E.coli?

Answer 13

pET-system which uses
A pET-series of expression vectors
And a strain of E.coli known as BL21(DE3)

Question 14

pET-vectors contain a powerful T7 promoter. Why is this so important and powerful?

Answer 14

More powerful than the regular promoter found in E.coli (LacP) because it produces a large number of mRNAs per second. It is not naturally found in E.coli.

Question 15

Why is regulation of recombinant gene expression really important?

Answer 15

Regulation is key because if there is no regulation then there is no expression of high quantities of recombinant protein.
So that we can get a lot of cell growth and prevent the slowing of growth

Question 16

What does the multiple cloning section allow us to do?

Answer 16

Allows us to clone for any gene we like

Question 17

Why is the T7 RBS important?

Answer 17

Without it, there wouldn’t be any translation of the gene

Question 18

What are the 2 regualtory sites on the pET vector system in BL21(DE3)?

Answer 18

1) LacP on the BL21(DE3) host E.coli which stops T7 RNA pol transcription
2) T7 promoter on pET-vector which stops transcription of recombinant gene

Question 19

What is IPTG and why is it needed for recombinant protein production?

Answer 19

It is the lactose analog and inducer.

It is needed so that the repressor can be removed= T7 RNA pol produced= recombinant protein made.

Question 20

Even in the absence of IPTG, there can be some T7 RNA pol expression. What is this termed as?

Answer 20

Leaky expression which causes small amounts of toxic protein to be made causing lysis of the cells.

Question 21

How is leaky expression overcome?

Answer 21

By adding pLysS (another recombinant protein), producing T7 lysozyme which inactivates T7 RNA pol. This prevents toxic protein being made by the cells.

Question 22

What are the rules for designing PCR primers?

Answer 22

Must be >18 nt long
Must finish at the 3’ end in G or C
Tm values of the primer pairs must be within 5 degrees celcius of eachother
Annealing temperature is 5 degrees less than the lowest Tm value of the primer pair

Question 23

What is the forward primer complementary to?

What is the reverse primer complementary to?

Answer 23

Complementary to antisense strand

Complementary to sense strand

Question 24

What are the 2 restriction sites that are added to the forward and reverse primers?

Answer 24

NdeI (CAT ATG) for FP and XhoI (CTC GAG) for RP

Question 25

If you wanted to design primers in an N-terminally histidine tagged protein, what pet vector do you use and do you include the stop codon?

Answer 25

Use pET-28a vector and include the stop codon on the RP

Question 26

For a C-terminally tagged protein, do you include the stop codon and which vector do you use?

Answer 26

pET-22b vector and don’t include the stop codon on the RP

Question 27

For a recombinant protein with no tag, which vector is used and is the stop codon included?

Answer 27

pET-22b and yes include the stop codon

Question 28

Which annealing temperature is used for a PCR with 5 cycles?

Answer 28

Primary annealing temperature (before adding restriction sites)

Question 29

Which annealing temperature is used for a PCR with 20 or more cycles?

Answer 29

Secondary annealing temperature (after adding restriction sites)

Question 30

How are His-tagged recombinant proteins purified?

Answer 30

Using HisTrap equipment which works by metal chelate affinity chromatography. (what we did in the practical)

Question 31

How do histidine residues on the recombinant protein vector bind to metal ions (nickel) in the columns?

Answer 31

Binds by sharing its lone pair of electrons with electron deficient orbitals of the metal ion.

Question 32

What other factors (apart from RBS and promoters) are involved in the expression of recombinant genes in E.coli?

Answer 32

Insolubility of recombinant protein
mRNA stability
Codon utilization
Transcription termination

Question 33

What are inclusion bodies and why do they form?

Answer 33

They are insoluble clumps formed when some recombinant protein does not fold correctly when translated. This is because chaperones aren’t present to aid in the folding and because proteins are produced very quickly so less time for correct folding.

Question 34

How can inclusion bodies be reduced?

Answer 34

By reducing the temperature that E.coli grows in (giving more time for protein to fold)
By linking the recombinant protein to a soluble fusion protein.

Question 35

How is a recombinant protein linked to a fusion protein?

Answer 35

Put the recombinant protein into pET-32 vector if solubility is an issue. The trx gene in pET32 codes for a thioredoxin fusion protein and is linked to the recombinant protein by cloning the protein in the Trx tag region.

Question 36

What are the 4 RNAses that degrade RNA?

Answer 36

RNase 2
RNase 3
RNaseE
PNPase

Question 37

How can the problem of ‘limited translation’ in recombinant genes that contain codons which are not commonly used in E.coli be overcome?

Answer 37

Use BL21(DE3) Rosetta which supplies tRNAs for many different codons. Increasing translation of proteins.

Question 38

What is one specific problem associated with expressing eukaryotic genes in E.coli?

Answer 38

Introns must be removed from gene before cloning because E.coli has no introns

Post-translational modification is not possible in E.coli which is an issue if those modifications are needed for enzyme activity (so we use yeast or mammalian cells not E.coli)

Question 39

Why are transcription terminators important? (3 reasons)

Answer 39

Stops transcription of unnecessarily long transcripts
Stops undesirable secondary structures forming (like hairpin loops), saving energy
Stops promoter occlusion