Using DNA to make proteins - Professor Shephard

Question 1

What was the first protein to be expressed unnaturally?

Answer 1

Insulin, this was used for diabetes treatment. Nowadays all insulin used in the treatment of diabetes is recombinant insulin.

Question 2

How have researchers solved the issue of obtaining proteins that are difficult to purify or are made very rearely in the cell.

Answer 2

They can express the protein form its cDNA, cDNA is obtained through reverse transcribing mRNA using reverse transcriptase.

Question 3

What are some advantages of using cDNA to express a protein?

Answer 3

It is possible to obtain large amounts of your protein of interest and it is possible to produce a mutant protein by mutating the cDNA.

Question 4

What are some uses of expressed proteins?

Answer 4

• Production of substances useful for medicine or industry
• Studying structure and function of that protein
• Study effects of mutations on that protein
• Studying localisations and movements of that protein in a cell

Question 5

What are the three main expression systems used to produce large amounts of protein?

Answer 5

• Bacteria (E. coli)
• Insect Cells
• Mammalian Cells

Question 6

Describe the structure of cDNA used to express a protein of interest

Answer 6

cDNA is the sequence that codes for the protein, it does not contain any introns. The cDNA sequence will be designed to start with ATG because this is a translation initiation codon, it will have a T instead of a U as it is in cDNA at the moment. The cDNA sequence will be designed to end with TAA, TAG or TGA because these are all translation stop codons.

Question 7

How is an expression system chosen?

Answer 7

The choice depends on the protein you are trying to express, the best expression system for your particular protein of interest is usually found using a trail and error method.

Question 8

Why are inducible expression vectors (plasmids) used to express our protein of interest in bacteria?

Answer 8

So that we have the ability to regulate the protein production in the bacteria. If the expression is not regulated then the bacteria may make huge amounts of the foreign protein and this may be toxic to the bacteria and kill it, which is not what we want. Too much protein being expressed may also lead to inclusion bodies that will precipitate out in the bacteria and it is often impossible to re-solubilise the proteins.

Question 9

How do we regulate protein production in a bacteria on a inducible expression vector?

Answer 9

We place DNA that codes for our protein of interest downstream of an inducible promoter that can be turned off. This is to ensure that it is possible to switch on and off protein production in the bacteria to avoid overwhelming the bacteria and producing too much protein

Question 10

What are the key features of an inducible expression vector?

Answer 10

1. Promoter - this is where RNA polymerase can bind. The promoter region must be able to be recognised by bacterial RNA polymerases because the host polymerase will be transcribing the genes.
2. MCS (multiple cloning sites) - This is needed so that the gene of interest can be inserted into the vector using a restriction endonuclease
3. RBS (ribosomal binding sites) - This is needed so that once the mRNA is transcribed the ribosome can bind and begin to translate the protein
4. TER (transcription terminator) - We need this to stop the ribosome translating once the protein is made.

Question 11

What are the two most common promoter sequences seen in inducible expression vectors?

Answer 11

trc hybrid promoter and the T7 phage promoter.

Question 12

What are the features of the trc hybrid promoter?

Answer 12

trc hybrid promoter is a strong promoter that will bind to RNA polymerase effectively. The trc promoter has the -35 box from a trp promoter and the -10 box from the Lac promoter. The host RNA polymerase will recognise this promoter. The hybrid -35 and -10 boxes makes this promoter very strong. The hybrid trc promoter also contains the inducible Lac operator. IPTG can interact with the repressor to stop it from binding to the operator, preventing the gene from being switched off. IPTG induces protein expression, this gives another level of control of the production of your protein of interest.

Question 13

What are the features of the T7 phage promoter?

Answer 13

Host bacterial RNA polymerase does not recognise the T7 phage promoter so a second plasmid is used. This second plasmid contains the genes for the T7 RNA polymerase, the plasmid also contains a Lac promoter and operator. The Lac repressor is encoded on the chromosome of the bacteria. If IPTG is added, this binds to the Lac repressor and causes the Lac promoter to be switched on, therefore T7 RNA polymerase is produced. T7 RNA polymerase then binds to the promoter region on the first plasmid (the T7 phage promoter) and the cDNA is transcribed.

Question 14

What are the disadvantages of using bacteria to express eukaryotic proteins?

Answer 14

1. Lack of glycosylation - This is a very important step in terms of protein production, if a protein lacks these sugar groups we cannot be sure that its behaviour will be the same as the wild-type.
2. Eukaryotic protein expressed in bacteria are often insoluble and this leads to inclusion bodies.
3. Incorrect folding can occur due to the folding occurring in a different environment.
4. Little activity - Even if the expression system works well and lots of protein is made, you may get very little activity out of your protein.

Question 15

What are the advantages of using Baculovirus/ insect cell expression systems for eukaryotic proteins?

Answer 15

1. Correct protein folding - insect cell is a eukaryotic cell and so folding is likely to be correct
2. Sub-cellular targeting - insect is a eukaryotic cell so the protein is likely to be correctly targeted to a specific location.
3. Biological activity - Usually very high as it is being expressed in a eukaryotic cell
4. Post-translational processing - Bacterial cells cannot perform any post-translational modifications but a eukaryotic expression system can, these have huge effects on a proteins function
5. High yield - this type of expression system tends to give very high yields.

Question 16

What are the two most commonly used insect host expression systems?

Answer 16

Fall army worm (SF9)
Cabbage looper (Tni)

Question 17

What is the cloning vector used for insect cell expression systems?

Answer 17

Baculovirus, it is a virus that naturally infects insect cells. The virus is VERY big (130kb), therefore it is very difficult to manipulate in a test tube.

Question 18

Why is the transposition approach used in bacterial cells to clone foreign DNA into the baculovirus genome to be used in an insect cell?

Answer 18

Because the Baculovirus is so large, this is beneficial as you protect the size of the baculovirus genome as you are not cutting away at it with restriction enzymes.

Question 19

Describe the process of trying to get a DNA sequence into a Baculovirus without using restriction enzymes or DNA ligase? The transposition approach

Answer 19

1. Clone the cDNA of interest, this is done using a small plasmid. The small plasmid has been modified so that it has a promoter sequence (Polyhedrin promoter - POL H) that provides the DNA sequence that the host RNA polymerase form the insect cell can bind to. After binding the protein of interest will be produced. On either side of the promoter and foreign DNA sequence there are two transposition sequences called Tn7R and Tn7L.
2. The small plasmid is transformed into competent DH10BAC E. coli cells. The bacterial genome genome has been modified so as well as the Baculovirus genome it contains the LacZ gene and a attTn7 site so that transposition can occur. The molecule is called a Bacmid as it is a bacterial genome with a bit of a plasmid. An additional helper plasmid is also provided which encodes all the proteins that are needed to carry out the transposition.
3. Transposition occurs and the creation of the recombinant Bacmid and LacZ E. coli. This is so that the recombinant cells can be selected for on a blue white assay, white colonies form is it is recombinant as it has interupted the LacZ gene.
4. Isolate Bacmid DNA form the E .coli cell
5. Infect the insect cells. The bacmid contains all the genes to make the viruses and also our protein of interest.
6. Infect the cells with the virus particles. There will be recombinant protein expression and one of these proteins will be our protein of interest.

Question 20

What are the disadvantages of expressing proteins in mammalian cells?

Answer 20

1. Expensive to culture - eukaryotic cells have to be nurtured to ensure functionality and they require a lot of nutrients
2. Not easy to produce large amounts of protein

Question 21

What are the advantages of expressing protein in mammalian cells?

Answer 21

1. Correct folding usually occurs as you are expressing mammalian proteins in mammalian cells
2. Correct post-translational modifications
3. Correct biological activity
4. Correct sub-cellular targeting

Question 22

Why are shuttle vectors needed in mammalian expression systems?

Answer 22

It is always necessary to clone the DNA of interest first and then transform it into mammalian cells, this is where a shuttle vector is used.
Shuttle vectors can replicate in both bacterial cells (to clone the DNA) and in eukaryotic cells (to express the cloned DNA). A shuttle vector can move from bacterial to eukaryotic cells and can replicate in both. It is larger then a normal vector as it contains sequences that allow it to replicate in the two cell types. They contain MCS, antibiotic resistance genes and two origins of replication, one for each cell type.

Question 23

Describe both of the promoters on a shuttle vector

Answer 23

The shuttle vector will need to have a eukaryotic promoter upstream of the MCS that will only be recognised by a eukaryotic RNA polymerase. The vector also needs to encode for a polyA tail as the mRNA created will need this in the cell. The vector also has a second eukaryotic promoter with an associated antibiotic resistance gene to allow for selection, this resistance being controlled by a eukaryotic promoter means that the plasmid will only give resistance to a eukaryotic cell, this allows you to select for the plasmid in prokaryotes and eukaryotes.

Question 24

What is transient transfection?

Answer 24

Transfecting the plasmid into a mammalian cell. Lipid treatment is when the plasmid is coated in lipid so that it can pass through the plasma membrane. Electroporation is when the mammalian cells are given shocks that make them take up DNA. These selection events are needed as it is a rare event for a plasmid to go into the nucleus of a cell and start to be transcribed.

Question 25

What is a chimeric protein? Give an example of how chimeric proteins have been used in research

Answer 25

Chimeric proteins are when bits of DNA are joined together and expressed to create a protein that does not normally exist in a cell. Researchers created a chimeric protein by adding what they suspected to be the matrix targeting sequence to a cytosolic protein, they expressed it in a eukaryotic cell and saw where it went, indeed it went into the matrix.

Question 26

Describe the experiment involving DHFR protein

Answer 26

DHFR protein binds methotrexate inhibitor when it is in its folded state, this complex can also be tracked by researchers. They could use this to see if a protein is folded when it crosses the mitochondrial matrix, they found that folded protein bound to methotrexate inhibitor could not cross the membrane and so found out that proteins need to be in their unfolded state to cross the mitochondrial membrane.

Question 27

Describe how a shuttle vector can be modified to track protein movement in a eukaryotic cell

Answer 27

It is possible to track where a particular protein of interest goes in a cell by adding codons for fluorescent protein into the shuttle vector, the shuttle vector would transcribe your protein of interest, then the fluorescent domain and then the polyA tail. GFP is the most well known. The fusion protein produced that will fluoresce so you can track its movements.

Question 28

Describe how you would conduct an experiment to determine the effect of a mutation on protein function

Answer 28

Introduce a mutation in the cDNA of your protein of interest. Express the mutant protein. Compare function or normal and mutant proteins.

Question 29

What is protein engineering?

Answer 29

Changing a specific amino acid in a protein to create specific mutations in the DNA in order to compare its function to wild-type proteins.

Question 30

Describe the method of using oligonucleotide site directed mutagenesis to make a mutation in a specific area of the genome

Answer 30

1. Heat a double stranded plasmid to create single methylated strands and these are now the template mother strands
2. The mutagenic oligonucleotide primers are annealed. These primers are short molecules of about 20 nucleotides that can be made in a lab. The mutagenic primer contains the complementary sequence to the change you want to incorporate.
3. Extend the primer with DNA polymerase and copy the mother strand with proof reading DNA polymerase (Pfu)
4. Denature the plasmids and then re-anneal them. They will come together in different combinations.
5. The two newly synthesised strands coming together will form the double stranded plasmid with no methyl groups attached. This is also the plasmid that has the mutation in both strands
6. Add Dpn1 to digest the methylated mother strands. This enzyme will digest the mother strands and any hemi methylated strands. So the only molecule that it does not digest is the new strands that have incorporated the mutations.

Question 31

What is EMSA?

Answer 31

Electrophoretic mobility shift assay and it tests for DNA binding. If the protein does bind to its targets it will be a heavier complex and it will not move as far in the acrylamide gel. The DNA is labelled, therefore, it is possible to see if the mutation induced does effect the binding of the protein. This experiment is always done in an excess of probe.