Lecture 3 protein overexpression Flashcards
How can you overexpress a protein (or gene, actually)
- Construct a plasmid with the gene coding your protein
- Introduce this plasmid into a suitable organism (host)
- Have the protein synthesized by the host
Why would you want to overexpress a protein?
To produce a large amount of a protein(investigate, sell…) To introduce directed mutations into your protein
What does an overexpression system consist of?
- An over-expression host, suitable organism to produce the protein
- An expression vector, plasmid with the gene sequence for your protein
Examples of host organisms?
Bacteria (relatively easy...) Mammalian cells (e.g. human proteins) Yeast (simplest eucaryote) Insect cells (high level product..) Plants (plant proteins) Fungi (fungal proteins) Transgenic animals (complicated..)
How to plan your over-expression strategy?
Try with simplest system (bacteria, E. coli)
In case of post-translational modification: Yeast
If expression not satisfactory: Other hosts or in vitro
Help from company Handbooks(e.g. from GE Healthcare)
What do over-expression Vectors contain?
Plasmid with: Gene sequence for your protein
Strong promotor for mRNA production
Control of transcription
Other useful features (e.g. tag for purification or detection, protease sites)
What is an example of vectors that are often used for over-expression?
Example: pET vectors
- Often used for expression
- Tightly controlled
- Particularly useful
- for harmful proteins
- Many versions available
What is another example?
Example: pET-24a.
- Resistance to the antibiotic Kanamycin
- Expresses protein with Histidin-tag
How are cells made competent?
-> Cells have to be treated (made competent):
Chemically (usually calcium chloride)
Electrically (Electroporator)
How to grow bacteria regarding large scale expression?
Grow picked colonies of transformed bacteria in liquid growth medium: Start with small amount (~ 5 ml) check growth, expression. Then grow in larger volume (> 1l, depends on available facilities).
What if the expressed protein is harmful for host bacterium?
Induction:
Make two steps: 1.) Grow bacteria 2.) Start (induce) transcription of protein
-> Frequently used inducer: IPTG (iso-propyl-thio-galactoside)
What’s a fusion protein? Why to express fusion proteins? How to make a fusion protein?
What’s a fusion protein? Two proteins produced together as a single polypeptide chain
Why to express fusion proteins? 1.) For easy purification: Protein of interest + Protein easy to purify 2.) For easy detection: Protein of interest + Protein easy to detect
How to make a fusion protein? Combine DNA coding for both proteins by genetic engineering
Fusion Proteins: Tags. What is a tag? Name examples
Tag: Short sequence fused to protein of interest for easy purification:
Examples for Tags: Histidine-Tag (6-10 histidine residues interacting with nickel compounds)
Strept-Tag (short protein sequence interacting with Streptavidin)
What are two strategies for constructing a plasmid for a histidine-tag?
Two strategies:’
- ) Insert sequence coding for his-tag into gene coding for protein of interest
- ) Insert sequence coding for protein of interest into specialplasmids already containing sequence for his-tag
His-tag can be N-terminalor C-terminal
How to purify proteins with a his-tag?
- Disrupt cells
- Purify cell extract with affinity column (Nickel-Nitrilotriacetic acid, NTA)
- Binding of His-Tag (Nearly) Only proteins with His-tag bind!
- Elute bound protein with Imidazole (competing with histidine)
