Fergus - Vector Question Flashcards
List the advantages of bacterial vectors
(5)
- Large quantities of protein rapidly produced
- Cheap - need very cheap medium (LB broth)
- Quick expression and purification
- Often useful for antigens
- Possibly active proteins
List the disadvantages of bacterial vectors
(6)
- Poor post-translational capabilities
- Only cDNA can be used
- Protein folding can be incorrect
- Solubility of protein in bacterial cell can be poor
- Will not splice out introns
- Conditions in bacteria might be different -> different pH or salt concentrations
List the advantages of eukaryotic vectors
(6)
- Genomic clones acceptable
- Post-translational events generally occur
- Solubility generally good
- Stable cell lines can be generated -> will constantly produce proteins for us
- With appropriate signals we can get secrete the protein into cell culture medium possible -> we can purify from this
- 6xHis purification possible
List the disadvantages of eukaryotic vectors
(5)
- Low levels of expression
- Slow growth of cultures -> eukaryotic cells divide every 24 hours
- Expensive -> culture medium and equipment
- Purification is more difficult
- Cells can become contaminated with bacteria -> could waste a week trying to grow a culture
Give an example of a bacterial vector
pGex
Give an example of a eukaryotic vector
pCMV
List the components of pGex
(7)
Myc
Stop
Amp
Ori
Lac 1
Ptac
GST
What is Myc
Short segments of DNA which contain several restriction sites allowing for the easy insertion of DNA
What is Amp
Ampicillin resistance gene
Allow for selection of plasmid-containing bacteria
What is Ori
Origin of replication
DNA sequence which allows initiation of replication within a plasmid by recruiting transcriptional machinery proteins
What is Lac 1
Lac operon
Gene responsible for the production of our protein
What is Ptac
-> tac-promoter (lac operon promoter)
The region that drives transcription of the target gene
It determines which cell types the gene is expressed in and the amount of recombinant protein obtained
What is GST
glutathione S transferase
Gene that allows for protein purification
List the components of PCMV
(9)
MCS
T SV40
F1 ori
Pbla
Ori P SV40
Neo
TK poly A
PCMV
6x His
What is T SV40
o SV40 transcription terminator
o This has a poly A signal on it -> it will polyadenylate sequence (terminate)
What is F1 ori
o Filamentous phage origin of replication
o Allows generation of single stranded copy of the sequence (useful in sequencing)
What is Pbla
Amp
What is Ori P SV40
o Origin of replication
o Allows plasmids to replication in eukaryotic cells
What is neo
Neomycin
Confers resistance to G418 in eukaryotes
What is TK PolyA
o Thymidine kinase poly A signal
What is PCMV
o Cytomagalovirus promoter
o Induces high levels of expression of proteins in eukaryotic cells
What is 6xHis and how does it work
o protein purification
o 6 histadines in a row
o This does not exist in nature (eukaryotes have a max of 3 in a row, bacteria barely have any histadines)
o 6x Histadine is tagged onto our protein
o 6x His Fusion protein results
o 6X His binds to Nc++ (nickel atom)
o 6x His will binds to Nickel agarose bead
o Retention of 6x His (bound to protein) in the column
o Wash everything else away
o Add excess nickel to compete with the protein for binding
o This will give us our purified proteins
Write a note on recombinant DNA
(5)
- DNA that has been formed artificially by combining constituents from different organisms
- Recombinant DNA is made by breaking up two sequences of DNA using an endonuclease e.g. HaeIII
- HaeIII cuts at
o 5’ GGCC 3’
o 3’ CCGG 5’ - HaeIII creates two blunt ends which will join together in the presence of DNA ligase to create recombinant DNA
- Other nucleases such as EcoR1 create sticky ends which will also join together in the presence of DNA ligase
When asked about producing recombinant DNA using a bacterial host, what headings do you use?
(5)
Recombinant DNA
Creating recombinant DNA
Bacterial transformation
Protein Expression
Protein purification
Write a note on creating recombinant DNA
(5)
We amplify the insulin gene using PCR
The gene is amplified in such a way that restriction enzyme sites which complement pGex are incorporated
The insulin gene is also combined with the gene for GST
We cut the PCR product and pGex using Xho 1 and Eco R1 restriction enzymes
We mix the two products together in the presence of T4 DNA ligase - sticky ends combine to form recombinant DNA incorporated in a pGex plasmid
Write a note on protein expression
We induce protein (insulin) expression by pGex by using IPTG (a form of lactose that bacteria cannot metabolise)
IPTG triggers the transcription of the lac operon and this induces protein expression
Write a note on bacterial transformation
(3)
Uptake of plasmids by E.Coli BL21
By means of chemical treatment (CaCl2) and heat shock or electroporation and low salt conditions
Transformed cells are then selected using antibiotic resistance
Write a note on protein purification
(6)
GST method of purifying proteins
GST is not normally found in bacteria
We combine the gene for GST and our cDNA (gene of protein we want to express)
A fusion protein results - part is GST and the other is our needed protein e.g. insulin
Purification on glutathione sepharose beads -> fusion protein binds to these beads
Cleave off the GST from the protein
Pure protein results
