Protein engineering 2 Flashcards
What is screening?
Assaying all mutants for desirable improved qualities
What is selection?
Initially perform a step that removes the inactive proteins
before screening
What are the two types of selection?
Biochemical selection
Selection for binding
What is biochemical selection?
A cell will die and not form a colony without being transformed
What is the sequence space problem?
It is not possible to store billions of DNA sequences
What type of screening works well for hydrolytic enzymes?
Colorimetric or fluorescence detection
Can hydrolyse fluorescent substrates allowing them to fluoresce and changes to be detected
What is an example of a Colorimetric or fluorescence detection for hydrolytic enzymes?
O-Nitrophenol (Absorbance: 400-420nm, turns yellow when the group is released) Methyl Umbelliferone (Fluorescence: Ex 365 nm, Em 455 nm)
How is pipetting of cells into wells sped up?
Robots
What methods sorts 1000-2000 cells a second by levels of fluorescence?
Flow cytometry
What changes which way a cell will fall in flow cytometry?
Electronic pules in response to levels of fluorescence
In flow cytometry what do you do if the fluorescence is secreted?
coat the cell to encapsulate the fluorescence
What is microfluidics?
Miniaturized lab processes (that involve fluids)
e.g. flow cytometry on a small scale
In microfluidics does every oil droplet contain a cell?
No, some droplets will not contain cells.
Droplets are introduced more frequently than cells to prevent more than one cell being in a droplet
What is the primary microfluidics?
Cheap and fast
What is selection by display?
Display the library on something so that you there is a link between phenotype and genotype
How does phage display work?
Fuse library with p3 gene (protein coat) so that the POI is expressed on the surface of the phage
These can then be used for binding assays
What are the advantages of phage display?
fusion doesn’t affect infectivity so can increase population with E.coli, stable particles, high rate of phage synthesis
What is the disadvantage of phage display?
Because the protein is propagated in E.coli it is biased towards proteins that are non-toxic to E.coli
What is bacteria display?
Fuse protein to an outer membrane protein (e.g. ompA in E.coli)
These are then assayed for binding
If your protein is small where might you insert it in bacterial display?
In a loop region of a surface protein
If your protein is large where might you insert it in bacterial display?
Inserted terminally which may require a truncation to achieve
Why might you choose bacterial display over phage display?
Phage display requires one extra step because you have to propagate library in E coli
Lower risk
What is the primary advantage of yeast surface display?
Convenient for proteins that requires eukaryotic PTM’s for folding
How does yeast display work?
Normally fuse Aga2p to the protein
Aga1p is naturally on the surface of the protein
Aga2p is secreted and binds to Aga1 via disulphide bonds
A reducing agent can therefore be used to elute the Aga2-POI
How might you visualise whether the POI is expressed on the surface and whether it is able to interact with the binding partner?
Tag the binding partner (maybe with fluorescently tagged avidin)
Tag the POI (maybe with GFP)
In this example high green and low red would show lots of POI expressed but binding partners not able to bind
What is ribosome display?
Library with no stop codon is subjected to transcription and translation
The protein never disengages with the ribosome
Hence protein, mRNA and ribosome is all captured together
Selection, elution, reverse transcription, repeat
What are the advantages of ribosome display?
Not limited by transformation efficiency, no toxicity bias, ~1 day per cycle
What are the disadvantages of ribosome display?
No PTM, mRNA not very stable – lots of RNases on skin