Föreläsning 3: Production of heterologous proteins in bacteria and yeast

What is a heterologous protein

Protein that is produced by a host organism but originates from a different species

How do make a recombinant protein (general steps)

1. Gene encoding the protein of interest is isolated from an animal cell

2. It’s inserted into a cloning vector

3. The vector is introduced into a host organism

4. The host transcribes and translates the gene → protein

What are the key challenges producing a recombinant protein

• Which cloning vector: must be compatible and efficient

• How to get high protein yield

• Which organism to express the protein in: depends on the complexity of the protein (post-translational modifications)

Recombinant host

Is inserted with the gene and produces the wanted protein.

Why is often E. coli used for production of recombinant proteins

• Fast-growing

• Easy to manipulate

• High yields

• Backed by decades of research

→ perfect for simple protein expression

Why use other hosts

• For more complex proteins

• Some proteins require post-translational modifications (like glycosylation) that bacteria can't perform

• Choice of host depends on the type and complexity of the protein.

Why can’t we just put the gene into any plasmid?

A normal plasmid (eukaryotic) doesn’t have the right signals for E. coli to recognize and use the foreign gene. Instead, a special “expression vector” — a plasmid designed to make E. coli produce protein, is used. The expression vectors contain 3 important signals surrounding the gene:

• P: promoter

• R: ribosome binding site

• T: terminator

Together these form the expression cassette where the foreign gene is inserted.

Promoter (P)

• Tells the cell where to start making RNA and determines the rate of mRNA synthesis. More mRNA → more recombinant protein.

• A strong promoter is needed to produce much protein.

• Eukaryotic promoters (like the TATA box at -25) are not recognized by E. coli RNA polymerase, which instead needs bacterial promoter sequences like the -35 (TTGACA) and -10 (TATAAT) boxes.

• → To produce a eukaryotic (foreign) protein in E. coli, the gene must be placed under a bacterial promoter that E. coli can read and transcribe efficiently

Ribosome bindning site (R) (

Where ribosome attaches to mRNA

Terminator (T)

Stops DNA transcription (stem loop)

Why do we need regulation of promoters

Sometimes the protein can be toxic or use too much energy, so we don’t want it made all the time.

Describe regulatable promoters

• Inducible: Protein is made only when you add a chemical like IPTG. Gene is switched on when chemical is added

• Repressible: Protein is made unless a chemical turns it off. 

  Gene is switched off when chemical is added

Give some examples on promoters

• lac: induced by IPTG

• Trp: repressed by tryptophan

• Tac (hybrid of lac & trp): induced by IPTG

• λ: controlled by temp (turns on above 30°C)

• T7: IPTG + T7 RNA polymeras

Why do some cassettes sometimes include fusion between E. coli foreign gene

Sometimes the foreign gene is hard to express by itself → fusion

The benefits are:

• Avoiding weird secondary structure

• Avoiding the gene from being degraded by the bacteria

• Can act as signal peptide

• Help for purification

There are 2 main roots that problems with producing heterologous proteins in E. coli can derive from. What are they?

1. Problems due to the sequence of the foreign gene

2. Problems Caused by the Host — E. coli Itself

There are three problems caused by the sequence of the foreign gene. Name them and their solutions

1. Foreign genes have introns:

   • Eukaryotic genes often contain introns (non-coding regions), but E. coli lacks the machinery to remove them.

   • As a result, introns stay in the mRNA, leading to incorrect or non-functional proteins.

   • Solution: Use cDNA (complementary DNA) made from spliced mRNA (which has no introns), allowing correct expression in E. coli

2. The foreign gene contains E. coli termination signals:

   • Not all of the gene is transcribed

   • Solution: Use in vitro mutagenesis to remove or alter the termination-like sequences without affecting the protein’s amino acid sequence. In vitro mutagenesis =  introducing specific mutations into a DNA sequence in a test tube (outside of a living organism)

3. The foreign gene contains codon bias not ideal for E. coli

   • Different organisms prefer different codons for the same amino acid.

   • Solution use artificial gene synthesis to replace rare codons with those preferred by E. coli, optimizing translation without changing the protein.

There are three problems caused by the host cell. Name them and their solutions

1. Post-translational modifications are not done correctly (e.g glycosylation):

   • Some proteins require modifications like glycosylation after translation. E. coli cannot perform these eukaryotic-specific modifications.

   • Solution: use mutant E. coli strains engineered to perform limited modifications, though this is technically difficult.

2. Incorrect protein folding

   • Many eukaryotic proteins need help folding or forming disulfide bonds.

   • In E. coli, this can lead to misfolded proteins or insoluble aggregates (inclusion bodies).

   • Solution:  Engineer E. coli to produce chaperone proteins that assist in correct folding.

3. Protein degradation:

   • E. coli may degrade foreign proteins using its own proteases

   • Solution: Use mutant strains of E. coli that lack certain proteases to prevent unwanted degradation.

What is the workflow for constructing production strain

Production strain =  a specially engineered microorganism  (like E. coli) that has been modified to produce a specific protein or product in large amounts.

1. Choosing a vector

2. Designing the expression vector

3. Constructing the plasmid

4. Transformation and selection

5. Producing the protein

6. Purification and analysis

7. Evaluate the result

1. Choosing a vector

Decide what type of vector to use to carry the gene:

• Plasmid:

  • small circular DNA that replicate independently

  • Easy to manipulate

  • High copy number → gives more protein per cell

• Episomes (integrative vectors):

  • Integrate into bacterial chromosome

  • lower copy number but more genetically stable

  • Useful when long-term, stable expression is more important than quantity

2. Designing the Expression Vector

The vector is built by inserting the foreign gene into a complete expression system. The vector must contain:

• A strong promotor that works in (in E. coli: lac, T7)

• A ribosome binding site so the protein can be translated

• A terminator to end the transcription

• A selectable marker so you can identify the cells that have taken up the plasmid (e.g antibiotic resistance gene)

• An origin of replication (ori) so the plasmid can multiply inside the cell

• Optional: fusion tags (like His-tag) to help with purification, or signal peptides for protein export

3. Constructing the Plasmid

• Restriction enzymes cut the DNA

• Ligation joins the gene and vector

• Check that everything is correct, often using gel electrophoresis (size) or sequencing

4. Transformation and selection

You introduce the finished plasmid into the host. Not all cells will take up the expression vector → selection (e.g grow cells on a plate with antibiotics to exclude the unwanted cells):

• Only the bacteria that have taken up the plasmid (and the resistance gene) will survive

• These are the candidate production strain

5. Producing the Protein

Selected bacteria are grown in liquid culture under controlled conditions. You can:

• Add IPTG or another inducer to trigger expression.

• Monitor temperature, pH, and oxygen for optimal growth.

6. Purification and analysis

To isolate the protein, you:

• Lyse the cells (break them open).

• Use chromatography (often with fusion tags) to purify the protein.

• Use analysis methods like SDS-PAGE or activity assays to check:

  • Is the protein pure?

  • Is it the right size?

  • Is it active?

7. Evaluate the Result

Once the protein is purified, you ask:

• Is the purity good enough?

• What is the yield (g protein/g substrate)?

• How productive is the strain (g/Lh)?

• Is the process stable (does it work the same every time)?

What is the advantage of using yeast and filamentous fungi for production of heterologous proteins

• Yeasts are eukaryotes so closer to animals cells →  can process and fold animal proteins more efficiently, especially when the recombinant protein originates from animals

• You still need expression vectors to insert and express foreign genes.

What is the advantage of using  Saccharomyces cerevisiae for production of heterologous proteins

• A commonly used yeast species in biotechnology and brewing.

• It contains a natural plasmid called the 2 µm plasmid, which is:

  • Good size: 6.3 kb 

  • good copy number: 60 copies per cell.

What is the disadvantage of the 2 µm plasmid

• Has weak selectable markers (less efficient for selecting transformed cells)

Solution:

• A special strain of S. cerevisiae that lacks the LEU2 gene cannot make leucine and needs it supplied in the medium.

• LEU2 gene can act as selectable marker - organisms with the gene can survive without leucine

• If you insert the correct LEU2 gene into the plasmid, only transformed cells will survive on leucine-free medium.

So, LEU2 acts as a selectable marker: only cells that took up the plasmid can grow.

What is a shuttle vector

Plasmids that can function (replicate) in two different organisms

Describe yeast episomal plasmid (YEps)

• Plasmids that derive from the 2 µm plasmid are called YEps

• They are shuttle vectors: can function in both E. coli and S. cerevisiae

• Usually for shuttle vectors, you build and amplify the plasmid in E. coli (easy to grow), then transfer it into yeast for protein production

• Selective markers in shuttle vectors: ampR, LEU2, tetR.

How do you build and amplify the plasmid in E. coli and then transfer it to S. cerevisiae

• Du klonar eller bygger plasmiden i E. coli.

• ampR används här för att välja ut bakterier som har tagit upp plasmiden (de överlever på ampicillinplattor).

• Du får många kopior av plasmiden genom att odla E. coli → enkel och billig DNA-produktion.

Andra steget: in i jäst

• Sedan renar du plasmid-DNA:t från E. coli.

• Det renade plasmid-DNA:t förs sedan in i jästceller (t.ex. med kemisk behandling eller elektroporering).

• LEU2 används nu för att välja ut jästceller som tagit upp plasmiden – de överlever på leucin-fritt medium.

Alltså: pro

Name different yeast vectors

• YIps (Yeast Integrative plasmids)

• YRps (Yeast replicative plasmids)

• YCps (Yeast centromeric plasmids)

Describe YIps

• Must integrate into the yeast genome

• Very stable, but only one copy per cell

Describe YRps

• Have yeast origin of replication

• More copies per cell but less stable

Describe YCps

• Include a centromere sequence

• Stable but one copy per cell

What is yeast artificial chromosome (YAC)

• The YAC is the largest cloning vector used in yeast (can carry up to 1000 kb of DNA).

• It includes key parts of a chromosome: telomeres, centromere, and origin of replication.

• Useful for cloning very large pieces of DNA, like entire genes or genomic fragments.
  

What is the problem of using yeast instead of E.coli

The proteins produced by yeast are hyperglycosylated - get too many sugar chains added. This alters the protein structure and can reduce its function in humans.

What is the advantages of using S. cerevisiae

• High yield

• Safe

• Well studied

Name other useful yeast and filamentous fungi

• Pichia pastoris: can produce very high amounts of protein (up to 30% of total cell protein)

• Aspergillus niger: Secretes proteins directly into the medium, which makes purification easier.

Describe using insect cells

These cells (e.g., from Drosophila) are widely used because they have strong expression systems. This leads to high yields of recombinant protein, especially when using baculoviruses as vectors. Baculoviruses are viruses that naturally infect insect cells and are modified to carry the gene of interest.

Describe using mammalian cells

These provide the most accurate environment for producing human or other animal proteins. They perform complex post-translational modifications like glycosylation exactly as they would happen in the body. However, mammalian systems are expensive and slow, and they often result in lower protein yields.

Describe the genetic tools in mammalian systems

• Cloning vectors are not necessary but can boost the yield especially if strong promotor

• Viruses like SV40 can serve as cloning vectors.

• Microinjection: directly injecting the DNA in the nucleus of a cell, often in a fertilized egg to create transgenic animals

• Pharming = protein production in live animals

  • A common approach is to insert the gene into the milk-producing genes (e.g., using the β-lactoglobulin promoter).

  • This way, the protein is secreted in the milk and can be collected and purified easily.

What are the advantages and disadvantages of the different host systems