E. coli expression systems

Question 1

5 reasons to express in E.coli

Answer 1

• Well established system
• Easy to manipulate
• Large variety of Vectors, strains, methods
• Low-tech, safe and inexpensive
• Suitable for variety of labellings

Question 2

Why is E.coli easy to manipulate ?

Answer 2

• Plasmids, gene knockout systems, and transformation methods are well optimized for E. coli
• Easy to insert, delete, or modify genes using common methods like restriction cloning, Gibson assembly, or CRISPR
• High transformation efficiency allows rapid generation of recombinant strains.

Question 3

Give examples of labelling techniques that are used in E.coli

Answer 3

• Isotopic labellings : Incorporation of isotopes like 13C, 15N, or 2H for probing protein structures
• Non-natural amino acids : Selenomethionine (Se-Met) is used for phase determination in X-ray crystallography
• Radioactive labeling: Incorporation of isotopes like 35S, 14C, or 3H for biochemical assays.

Question 4

What are other more complex expression systems ? and when are they used?

Answer 4

Yeast
Insect cells
Mammalian cells

Used when E.coli has failed to yield a protein of desired quality

Question 5

What does expression system choice depend on ?

Answer 5

• Protein Complexity: Need for PTMs or proper folding.
• Cost and Speed: Budget and time constraints.
• Scale: Small-scale lab experiments vs industrial production.
• Application: Research, therapeutics, or industrial use.

Question 6

What’s missing in E.coli as an expression system ? 2 points

Answer 6

• no PTMs : glycolisation, disulfide bridges, lipidation, proteolytic processing
• Problems in solubility : missing chaperones, high level speed of expression -> Incorrect folding

Question 7

What are the elements of an expression vector ?

Answer 7

• Expression cassette
• Antibiotic resistance gene
• Origin of replication
• Other control genes

Question 8

What does an expression cassette contain ? 5

Answer 8

• Promoter
• Regulator binding site
• Ribosome binding site
• Multiple cloning site (MCS)
• transcriptor terminator

Question 9

What’s a Regulator Binding Site?

Answer 9

• The lac operator is a DNA sequence where the LacI repressor protein binds to block transcription in the absence of an inducer (e.g., IPTG).
• Enables tight control over expression to prevent leaky expression (toxic proteins)

Question 10

What’s a Ribosome Binding Site (RBS)?

Answer 10

A sequence upstream of the start codon that facilitates binding of ribosomes to initiate translation.
Ensures efficient translation of the mRNA into protein.

Question 11

What’s a Multiple Cloning Site (MCS)?

Answer 11

A short DNA sequence containing multiple unique restriction sites (e.g., EcoRI, BamHI, HindIII) for inserting the gene of interest.
Provides flexibility for cloning.

Question 12

What’s a Transcription Terminator ?

Answer 12

Stops transcription by providing a signal to RNA polymerase to detach from the DNA template.
Prevents readthrough into other parts of the plasmid.

Question 13

What’s an Origin of Replication (ori)?

Answer 13

A DNA sequence where replication begins, allowing the plasmid to be copied within the host cell.
Example: pBR322 ori ensures high plasmid copy numbers in E. coli.

Question 14

What’s a promoter?

Answer 14

A promoter is a DNA sequence located upstream of a gene that regulates the initiation of transcription by RNA polymerase. It acts as a binding site for RNA polymerase and associated transcription factors, determining when, where, and how much of a gene is expressed.

Question 15

What are the characteristics of prokaryotic promoters?

Answer 15

-35 box: Recognized by the sigma factor of RNA polymerase.

-10 box (Pribnow box): Facilitates unwinding of DNA.
Example: T7 promoter, used in bacterial expression systems.

Question 16

What are the 2 types of promoters?

Answer 16

• Inducible promoters : Activated only in the presence of a specific inducer, allowing temporal control of gene expression
• Constitutive Promoters: Continuously active, driving constant gene expression.

Question 17

Give me 4 functions of promoters ?

Answer 17

• Initiation of transcription
• Regulation of gene expression
• Specificity
• Scalability (strong promoters)

Question 18

3 categories of promoters used in E.coli vectors.

Answer 18

• E.coli natives : lac, trp, tac, trc, ara
• Viral but recognised by E.coli : T5
• T7, T7lac : requires its own RNA polymerase

Question 19

3 facts about the lac promoters

Answer 19

• promoter of the lac operon
• repressed by lacI gene, which binds downstream of promoter
• regulated by galactose or its analogues, in expression work non-hydrolysable IPTG used

Question 20

2 facts about trp promoters

Answer 20

• promoter of tryptophane biosynthetic enzymes
• respressed by Trp, so induction done by causing a Trp deficiency with indole-2-acrylic acid

Question 21

tac and trc systems (combo of trp and lac)

Answer 21

Synthetic promoters created by fusion of trp and lac promoters
- -35 part from trp, -10 from lac
- regulation from lac system
- much stronger than trp and lac

Question 22

4 facts about the araB promoter ?

Answer 22

• Promoter of the arabinose operon in E.coli
• very well repressed prior to induction
• Expression induced with arabinose
• Linearly tunable expression level (fine-tuning of expression levels by adjusting the concentration of arabinose in the medium)
• pBAD series vectors

Question 23

6 facts of the T7 system

Answer 23

• promoter of the gene1 of the bacteriophage T7
• Recognised only by the T7 RNA polymerase (faster enzyme)
• pET series of vectors
• T7RP can be inhibited by T7 lysozome (pLysS/E plasmids)
• combined with lacO regulator and lacI gene to provide tight regulation
• needs to be combined with a T7 transcription terminator

Question 24

How tight is the T7 system? 3 points

Answer 24

• LacI repressor prevents transcription from the lacUV5 promoter and production of T7RP. Sam repressor binds downstream of T7 promoter to prevent transcription in case T7RP is produced.
• T7 Lysozome can be expressed from a separate plasmid (pLysS or pLysE) to inhibit the polymerase
• Expression can be done in strain with no T7RP gene (introduction by sigma or M13 phage infection) as last resort

Question 25

How is T7 induced?

Answer 25

- IPTG binds to lac repressor releasing it from DNA. - Repression of the lacUV5 is released and T7RP is produced and induces transcription and expression

Question 26

5 examples of commercial vector families

Answer 26

- pET from Novagen : T7 promoter, lots of variants - pGEX from Amersham : original GST fusons, trc promoter -pQE from QIAGEN : T5 promoter, original His-tag vectors - pBAD from Invitrogen : ara promoter - pMAL from NEB: maltose binding protein fusions

Question 27

What's the BL21(DE3) strain?

Answer 27

- Original expression strain for the T7 promoter - carries DE3 lysogen phage which contains a copy of the T7RP under control of lacU5 - wild strain and grows fast - defective in OmpT(outer membrane protease that can degrade recombinant proteins) - Defective in lon protease ( Degrades misfolded or aggregated proteins)

Question 28

Give examples of redox modified strains (allow disulfide formation in the cytoplasm).

Answer 28

- AD494(DE3), BL21(DE3)trxB : trxB deletions - Origami(DE3), OrigamiB(DE3) : trxB, gor (glutathione oxidoreductase) deletions

Question 29

Give examples of rec- strains.

Answer 29

- BLR(DE3) : rec- BL21 - JM109(DE3) - HMS174 (DE3) (strains with inhibited recombination events = more stable proteins)

Question 30

Why is production of proteins containing disulfide bonds is problematic in E.coli ?

Answer 30

Cytoplasm of the bacteria is reducing and formation of disulfide bonds is unlikely, if formed stability is very low Strains with more oxidizing intracellular environment have been developed (redox modified strains) although not super useful

Question 31

6 conditions for a good expression vectors.

Answer 31

- Make sense structurally - be in frame with ATG codon(methionine) and/or any fusion - be in frame with the stop codon (avoid excessive tails) - contain suitable ends for cloning - correct in sequence - be compatible with the restriction sites you plan to use

Question 32

N-terminal or C-terminal fusions ?

Answer 32

Question 33

Considerations when cloning an insert..

Answer 33

- Correct Orientation: Ensure the insert is in the right orientation and reading frame in the vector. - Start Codon: Add an ATG start codon if there’s no N-terminal fusion. - Stop Codon: Include a stop codon in your PCR primer to avoid overly long proteins. - C-terminal Fusion: Align the reading frame properly to ensure correct fusion with the C-terminal tag. - Restriction Map: Create a restriction map to check compatibility between the insert and the vector. - Sequence Verification: Always sequence the final construct to confirm no errors from PCR or cloning. - Avoid Cutting Domains: Don’t cut in the middle of a functional domain or secondary structure. - Use Linkers: Identify unstructured regions to place linkers or for fusions. - Predict Structures: Use tools like SMART, Pfam, or JPred to analyze domains and structure. - Test Multiple Designs: Create several constructs with varying N- and C-terminal positions for better chances of success. - Automate for Scale: Use robotics or high-throughput systems for large-scale construct testing.

Question 34

3 tips of codon usage in construct design.

Answer 34

- Avoid use of rare codons when expressing heterologous proteins (tRNAs in E.coli scarse) : AGA/AGG, CCC, CTA, ATA, GGA - N-terminal codons are most important - Optimisation of 6-10 first codons

Question 35

4 must-haves of the 5'PCR primer at N-terminus.

Answer 35

- Restriction enzyme recognition site - ATG codon in frame followed by small aa to allow for its removal - Sequence that anneals with the target gene - Optimised codons for E.coli

Question 36

4 must-haves of the 5'PCR primer at N-terminus.

Answer 36

- Restriction enzyme recognition site - STOP codon TAA/TGA/TGA in frame with gene - Sequence that anneals with target gene - Remember to reverse complement

Question 37

What are 3 typical problems of expression ?

Answer 37

- Low or no expression : codon usafe, instability of RNA and/or protein - Degradation or premature termination - Insolubility : inclusion body formation

Question 38

Effect of rare codons on expression.

Answer 38

- slow translation and reduce protein yield, especially if they cluster together -Clusters of rare codons are more likely to stall the ribosome during translation due to insufficient tRNAs. - Tandem rare codons require back-to-back availability of scarce tRNAs, which is harder for the cell to achieve - Rare tRNAs may get substituted by other tRNAs due to limited availability: heterogeneity in the protein, which can affect its function and structural integrity. - Variation in the amino acid sequence due to tRNA mismatches can result in a mixture of protein isoforms : affects protein purity

Question 39

5 tips to fix degradation problems.

Answer 39

- Minimise expression time : do a time course of expression, up to 3h at 37°C, ON at 15°c. - Keep samples chilled as soon as expression is finished - Try a protease deficient strain - Use protease inhibitors at all stages - Make another construct

Question 40

Why are the T7 promoters very difficult to control?

Answer 40

Lactose permease is the product of the lacY gene and actively transports IPTG into the cells, making it hard to control the inducer levels. Using strains without the lacY gene (e.g., Tuner(DE3)) so IPTG enters the cells passively through diffusion.