A fusion protein is a protein that contains multiple protein/polypeptide sequences that are covalently attached to form one protein product.
What are features of an expression vector? (GST::CaMPARI sample)
promoter in the right space to drive transcription
start codon to be used as the ribosone for translation
GST tag
CaMPARI gene
stop codon
In the pET41a-CaMPARI vector as displayed on this slide, is the GST tag located upstream from CaMPARI or downstream from CaMPARI?
A) Upstream
B) Downstream
A) Upstream
the flow of the stream is in the direction of transcription. So since
GST is transcribed before CaMPARI, GST is located upstream from
CaMPARI. The promoter, start codon, and GST tag are all located upstream from CaMPARI
Advantages of making a fusion protein
1.One protein can be used to tag another protein for purification (e.g. attaching GST) through affinity chromatography.
2.One protein can be used to tag another protein for visualization (e.g. attaching GFP).
3.One protein can make another protein more stable/soluble – allow for purification and biochemical analysis.
Disadvantages of fusion proteins:
1.Fusion may result in altered folding - this will negatively impact function.
2.Fusion can change the transport location within the protein.
3.Fusion changes the immunological properties of the protein - antibodies will recognize both parts of a fusion protein.
What is the purpose of making GST:CaMPARI Fusion Protein?
We want to be able to purify it via affinity chromatography.
What properties of CaMPARI could be affected by the fusion with GST?(check all that apply)
[ ]Ability to bind calcium ions
[ ]Ability to be transcribed
[ ]Ability to photoconvert
[ ]Ability to fluoresce
[X]Ability to bind calcium ions
Correct, GST could cause the calmodulin domain of CaMPARI to improperly fold, which would interfere with calcium binding.
[ ]Ability to be transcribed
[X]Ability to photoconvert
GST could interfere with CaMPARI's ability to be photoconverted if GST causes CaMPARI to be misfolded.
[X]Ability to fluoresce
CaMPARI's fluorescent properties are dependent on protein
structure, so a GST fusion could destabilize that structure and
interfere with fluorescence.
How is the reading frame determined?
The reading frame is determined by the first start codon the ribosome encounters.
From which start codon (AUG) will the Ribosome start translating?
D) AUGUCU
Correct, is the first start codon located downstream of the RBS.
What can result from an Insertion/Deletion event?
Frameshift
A shift in the reading frame alters translation of the remaining codons, resulting in incorrect amino acids and/or stop codons
Which gene sequence is dictating the reading frame?
B) GST
Where do you determine when identifying the reading frame?
The start codon- ATG
When using primers to adjust reading frame in restriction cloning, extra nucleotides should be added in what location of the primer?
A) Between the restriction enzyme site and the primer homology region
Correct, this location will not interfere with the gene-specific
homolgous region annealing to your target area. Nucleotide here will
also not be removed after digestion with the restriction enzyme.
When do we need to consider reading frame?
C) when tagging either end of a protein
Whenever you want to produce a fusion protein you have to consider reading frame
Which of the following columns is most likely to occur?
D
What advantage does PCR screening offer?
Lets you gain information about your cloned plasmids with the strategic positioning of PCR primers
- We want information about both presence and orientation of the gene
What would you detect if you tried using the original amplification primers during PCR screening?
You would only get information about whether your gene was present
What is the "anchored PCR" strategy?
the primer (F) anneals to the vector sequence, primer (R) anneals to the insert
What is the result of PCR screening when the vector has insert in the incorrect orientation?
What is the result of PCR screening when the vector doesn't have the insert?
What is the result of PCR screening when the vector has the insert in the correct orientation?
What would the results of PCR screening be if you had multiple inserts of CaMPARI inserted into your vector?
B) A PCR product larger than the expected size for CaMPARI insertion alone
What type of cloning that we discussed in class has a basically 0% chance of multiple inserts being INCORRECTLY inserted into the vector?
B) Gibson Assembly cloning
Due to the requirements for long complementary sequences needed for
annealing and polymerase extension, the chance for multiple inserts in
Gibson Assembly is basically 0%.
Author
saucyocelot
ID
360986
Card Set
Biotech weeks 5-6
Description
Fusion proteins, reading frames, screening and Sanger Sequencing
