Recombinant Protein Expression Flashcards
Define recombinant protein.
Directed expression of a protein in a host cell in contrast to a native protein.
State five uses of recombinant proteins.
- Functional analysis.
- Structural analysis.
- Purification.
- Therapeutic.
- Biotechnology.
Define constitutive promoter.
Switched on all the time within the host cell.
Define inducible promoter.
Switched on in response to stimulus.
State six advantages of using prokaryotic genes in E. coli.
- Produce pure protein.
- Produce large quantities.
- Cheap.
- Absence of introns.
- Absence of contaminating enzymes.
- Higher expression level.
When using prokaryotic genes in E. coli, bacterial cells are transfected with a coding sequence for an enzyme. What is this enzyme?
DNA helicase.
When using prokaryotic genes in E. coli, what temperature is the viral promoter active at?
≥37°C
When using prokaryotic genes in E. coli, total cell protein is analysed by what?
SDS-PAGE (polyacrylamide gel electrophoresis)
When using prokaryotic genes in E. coli, the bacteria can be grown at 25°C. Will helicase be made?
No.
When using prokaryotic genes in E. coli, the bacteria can be grown at 42°C for two hours. Will helicase be made?
Yes, it becomes the most abundant protein species in the lysate.
When using prokaryotic genes in E. coli, what are the four enzyme recombinant products generated from DNA manipulation?
- Restriction enzymes.
- DNA ligases.
- RNA polymerases (in Taq polymerase).
- RNA polymerases.
What can be used to structurally analyse eukaryotic or prokaryotic genes?
X-ray crystallography.
When using X-ray crystallography, why is pure protein required?
In order to grow crystals.
When using X-ray crystallography, a narrow parallel beam of x-rays is directed at a well-ordered crystal. Some of the beams are scattered by the atoms in the crystal - what does this cause?
Scattered waves reinforce one another a certain points and appear as a pattern of diffraction spots.
When using X-ray crystallography, what is the diffraction pattern and peptide sequence used to produce?
An atomic model.
