Fergus - Vector Question

Question 1

List the advantages of bacterial vectors
(5)

Answer 1

• Large quantities of protein rapidly produced
• Cheap - need very cheap medium (LB broth)
• Quick expression and purification
• Often useful for antigens
• Possibly active proteins

Question 2

List the disadvantages of bacterial vectors
(6)

Answer 2

• 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

Question 3

List the advantages of eukaryotic vectors
(6)

Answer 3

• 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

Question 4

List the disadvantages of eukaryotic vectors
(5)

Answer 4

• 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

Question 5

Give an example of a bacterial vector

Answer 5

pGex

Question 6

Give an example of a eukaryotic vector

Answer 6

pCMV

Question 7

List the components of pGex
(7)

Answer 7

Myc
Stop
Amp
Ori
Lac 1
Ptac
GST

Question 8

What is Myc

Answer 8

 Short segments of DNA which contain several restriction sites allowing for the easy insertion of DNA

Question 9

What is Amp

Answer 9

Ampicillin resistance gene

 Allow for selection of plasmid-containing bacteria

Question 10

What is Ori

Answer 10

Origin of replication

 DNA sequence which allows initiation of replication within a plasmid by recruiting transcriptional machinery proteins

Question 11

What is Lac 1

Answer 11

Lac operon
 Gene responsible for the production of our protein

Question 12

What is Ptac

Answer 12

-> 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

Question 13

What is GST

Answer 13

glutathione S transferase
 Gene that allows for protein purification

Question 14

List the components of PCMV
(9)

Answer 14

MCS
T SV40
F1 ori
Pbla
Ori P SV40
Neo
TK poly A
PCMV
6x His

Question 15

What is T SV40

Answer 15

o SV40 transcription terminator
o This has a poly A signal on it -> it will polyadenylate sequence (terminate)

Question 16

What is F1 ori

Answer 16

o Filamentous phage origin of replication
o Allows generation of single stranded copy of the sequence (useful in sequencing)

Question 17

What is Pbla

Answer 17

Amp

Question 18

What is Ori P SV40

Answer 18

o Origin of replication
o Allows plasmids to replication in eukaryotic cells

Question 19

What is neo

Answer 19

Neomycin

Confers resistance to G418 in eukaryotes

Question 20

What is TK PolyA

Answer 20

o Thymidine kinase poly A signal

Question 21

What is PCMV

Answer 21

o Cytomagalovirus promoter
o Induces high levels of expression of proteins in eukaryotic cells

Question 22

What is 6xHis and how does it work

Answer 22

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

Question 23

Write a note on recombinant DNA
(5)

Answer 23

• 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

Question 24

When asked about producing recombinant DNA using a bacterial host, what headings do you use?
(5)

Answer 24

Recombinant DNA

Creating recombinant DNA

Bacterial transformation

Protein Expression

Protein purification

Question 25

Write a note on creating recombinant DNA
(5)

Answer 25

 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

Question 26

Write a note on protein expression

Answer 26

 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

Question 27

Write a note on bacterial transformation
(3)

Answer 27

 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

Question 28

Write a note on protein purification
(6)

Answer 28

 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