Recombinant Protein Expression Flashcards
What are the components of bacterial expression vectors
promoter, terminator, MCS, ribosome binding site and a sequence for fusion protein
What are the two commonly used oris
ColE1 and pUC
What are the features of ColE1
comes from Col E1 plasmid and low copy number
What are the features of the pUC
high copy number
What will happen if two different plasmids have the same ori in a cell?
They cannot be maintained stably over generations as they interfere with each others replication
How do inducible promoters work?
control expression level, add a regulatory chemical to induce gene expression
How do repressible promoters work?
When you add a regulatory chemical, the gene expression is repressed
What are the advantages of using these two types of promoters?
Control expression of gene products toxic to host organism and control time and duration of maximal protein expression
What are three commonly used promoters used for expression in E. Coli?
lac, trp, and tac
What does the T7 promoter do and what is required for its expression?
promoter of bacteriophage T7 DNA-dependent RNA polymerase and requires T7 RNAP for expression
How does the lac promoter work?
LacI is continually produced and binds to repressor sie until IPTG comes along binding to LacI, transcription can then continue, yield depends on how much ITPG you add
How does the trp promoter work?
Trp aporepressor binds 2 molecules of trp which inactivates transcription. IAA then comes along and binds tighter than trp and inactivates the repressor
How does the tac promoter work?
Trp and and lac mix, trp promoter but LacI binding site, induction using IPTG
How does the T7 promoter work?
T7 RNAP must be inserted into genome and is activated by ITPG
How does GST protein fusion work?
Glutathione is on agarose beads and placed in an elution column with fusion and GST. Wash with the buffer to remove unbound protein and elute with glutathione
What is the issue with GST?
Can alter protein function, needs to be cut with thrombin or factor Xa
How does His-tag fusion work?
6 histidines bind divalent metal ions, NTA is linked to surface of agarose beads and eluted with imidazole
How does GFP fusion work?
Fuse into live cell and visualize multiple sites at once, and can be targeted by antibodies in western blots
How do short polypeptide tags work?
bind to specific locations so it’s good for localization and purification and can be detected by antibodies
What are some factors affecting levels of protein expression?
promoter strength, plasmid copy number, protein stabiloty and recovery and codon bias (can be fixed by SDM)
Why is yeast used as a common organism for protein expression?
larger fragments can be cloned and they have a well established system because they are eukaryotes
What is a common promoter used in yeast and how does it work?
GAL1, induced by galactose which is converted to D-glucose 1-phosphate
What is the regulation of the GAL1 pathway?
GAL4 is a transcription activator for genes needed for the activation for galactose into glucose, GAL80 can either bind the inducer which activates GAL genes or GAL4 which means no activation of GAL genes
What are some examples of selectable markers for yeasts>
His3, Leu2, Trp1, Ura3
you would need to supply pyrimidines
What are the vectors used for mammalian expression in cells?
Plasmid and viral vectors
Why do we want to use viral vectors and what are some examples?
High transduction efficiency and strong levels of expression
adenovirus, lentivirus and baculovirus
What are the two oris used in mammalian expression
SV40 (simian virus 40) and BK/BPV (human BK or bovine papilomas virus)
What are some benefits and the drawback to using the SV40
very high copy number, good for transient expression, low frequency of stable transfection
What is the benefit and drawback to using the BK/BPV ori
low to moderate copy number but maintained autonomously and stably for a long time
What type of markers are used for mammalian expression?
dominant markers, normally drug resistance
What are the three common promoters for mammalian expression?
SV40, RSV LTR and CMV
What is the difference bewteen the SV40 and RSV LTR promoter?
RSV LTR is a long terminal repeat promoter
What is the regulation system used for mammalian expression and why?
Tet operon because high levels of IPTG needed to inhibit LacI is toxic to mammals
How does tet repression work?
Fusion of the DNA binding domain of TetR (repressor) with the activation of the HSP VP19 activation domain results in tTA, Tet binds tTA removing it from the promoter and turns off gene expression
How does the tet activation work?
tTA protein is modified so that its binding to promoter requires the presence of tet, and then it can bind promoter and turn it on
