Expression of recombinant proteins in E.coli Flashcards
Why do we express recombinant proteins in E.coli?
When E.coli divides, they will produce the produce the protein we want. Then we purify it out.
What is a recombinant gene often linked to so that it can be expressed in E.coli?
Linked to a PROMOTER that E.coli recognises.
Linked to a RIBOSOME BINDING SITE that E.coli recognises.
E.coli is not the only system we use to express recombinant proteins. What are some other ways?
Plant cells, mammalian cells, animal cells and yeast.
Name some protein based therapeutics.
Hormones, cytokines, vaccines and monoclonal antibodies.
The Lac Operon comprises of 3 structural genes. What are they?
LacZ- codes for B-galactosidase
LacY- codes for lactose permease
LacA- codes for transacetylase
The Lac Operon comprises of 3 regulatory genes. What are they?
LacP- promoter sequence
LacI- codes for the lac repressor gene
LacO- operator sequence
What is the function of B-galactosidase?
Hydrolyses the glycosidic bond between glucose and galactose (breaks down lactose)
What is the function of lactose permease?
Makes the cell permeable to lactose
What is the cis-acting sequence of the lac operon?
consists of LacP, LacO and the ribosome binding site
What is the trans-acting sequence of the lac operon?
LacI
A gene that produces something that has an effect away from the gene
What is the function of the lac-repressor?
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
What is an operon?
A sequence of genes working together for the same function/purpose
What is the most popular system used to express recombinant proteins in E.coli?
pET-system which uses
A pET-series of expression vectors
And a strain of E.coli known as BL21(DE3)
pET-vectors contain a powerful T7 promoter. Why is this so important and powerful?
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.
Why is regulation of recombinant gene expression really important?
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
What does the multiple cloning section allow us to do?
Allows us to clone for any gene we like
Why is the T7 RBS important?
Without it, there wouldn’t be any translation of the gene
What are the 2 regualtory sites on the pET vector system in BL21(DE3)?
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
What is IPTG and why is it needed for recombinant protein production?
It is the lactose analog and inducer.
It is needed so that the repressor can be removed= T7 RNA pol produced= recombinant protein made.
Even in the absence of IPTG, there can be some T7 RNA pol expression. What is this termed as?
Leaky expression which causes small amounts of toxic protein to be made causing lysis of the cells.
How is leaky expression overcome?
By adding pLysS (another recombinant protein), producing T7 lysozyme which inactivates T7 RNA pol. This prevents toxic protein being made by the cells.
What are the rules for designing PCR primers?
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
What is the forward primer complementary to?
What is the reverse primer complementary to?
Complementary to antisense strand
Complementary to sense strand
What are the 2 restriction sites that are added to the forward and reverse primers?
NdeI (CAT ATG) for FP and XhoI (CTC GAG) for RP
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?
Use pET-28a vector and include the stop codon on the RP
For a C-terminally tagged protein, do you include the stop codon and which vector do you use?
pET-22b vector and don’t include the stop codon on the RP
For a recombinant protein with no tag, which vector is used and is the stop codon included?
pET-22b and yes include the stop codon
Which annealing temperature is used for a PCR with 5 cycles?
Primary annealing temperature (before adding restriction sites)
Which annealing temperature is used for a PCR with 20 or more cycles?
Secondary annealing temperature (after adding restriction sites)
How are His-tagged recombinant proteins purified?
Using HisTrap equipment which works by metal chelate affinity chromatography. (what we did in the practical)
How do histidine residues on the recombinant protein vector bind to metal ions (nickel) in the columns?
Binds by sharing its lone pair of electrons with electron deficient orbitals of the metal ion.
What other factors (apart from RBS and promoters) are involved in the expression of recombinant genes in E.coli?
Insolubility of recombinant protein
mRNA stability
Codon utilization
Transcription termination
What are inclusion bodies and why do they form?
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.
How can inclusion bodies be reduced?
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.
How is a recombinant protein linked to a fusion protein?
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.
What are the 4 RNAses that degrade RNA?
RNase 2
RNase 3
RNaseE
PNPase
How can the problem of ‘limited translation’ in recombinant genes that contain codons which are not commonly used in E.coli be overcome?
Use BL21(DE3) Rosetta which supplies tRNAs for many different codons. Increasing translation of proteins.
What is one specific problem associated with expressing eukaryotic genes in E.coli?
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)
Why are transcription terminators important? (3 reasons)
Stops transcription of unnecessarily long transcripts
Stops undesirable secondary structures forming (like hairpin loops), saving energy
Stops promoter occlusion
