L25 - Protein-Protein interactions Flashcards
(14 cards)
define the interactome and what it shows
map of all molecular interactions within the cell
= all protein-protein interactions
= each dot represents a protein and the line shows what it inteacts with
knowing whata protein interacts with provdides insight into function and biological place
= ineractuons can occor at any point in acells life cycle or at different regions within cell
how could we use a map of virus-host protein interactions to identify drugs/therapiues
viruses hijack host cells protein machinery to all virus to move through and replicate in a cell
= identifying the proteins it interacts with may allow us to disrupt virus cycle and identify drug targets
describe relationship between number of proteins in complex and abundance
very few complexes with a large number of components
= many large complexes are transient = short lived inetarctions
what method do we use to map the ineractome
Tandem Affinity Purification (TAP)
describe the method of TAP with a TAP tag containg = calmodulin binding protein,TEV cleavage site and protein A - high affinity for IgG antibody
- modify a protein of interest with a TAP tag
- pass sample contating protein through IgG bead collumn
= protein of interst binds to beads due to protein A
- protein is released by cleavage in TEV site by protease
= removes the protein A bound to IgG bead
- run through Calmodulin bead collumn
= protein binds via CaM-bp to CaM
- EGTA used to bind to calcium and release protein complex
= 2 round of affinity purification = very pure
hgow many rounds of affinity purification are done in TAP - tandem affiniuty purification
2
- Protein A binds to IgG antibody beads
- Calmodulin binding protein binds to Calmodulilin in prescence of calcium
describe identifying proteins using MALDI (matrix-assisted laser desorption ionisation)- TOF (time of flight) Mass-spec
- protein of interst is bound to matrix containing trypsin and digested at lysine and arganine
- sample ionised by laser and use of electrical/magnetic fields cause peptides to ‘fly’ about
- refelctor lense accelerates movement projecting onto detector
= curve trajectory/TOF is different depending on mass/charge ratio
= peaks produced representing different peptdies –> height reflects abundance
describe yeast cells
simle eukaryotes
fast growing + can undergo post-tran modifications
why does antibiotic resistance not work as a well selction emthod for yeast
high number of mutations producing antibiotic-restance strains
= survival may not be due to transfected plasmid
what is used for selction methods in yeast instead of antibiotic resistance
Auxotrophic/nutrional selection = lacck of a particular nutrient
LacZ also used for blue/white screening –> B-galactosaidase breaks down XGAL
what do shuttle vectors contain
contain components for use in both bacteria and eukaryotes
- origins of replication for yeast AND bacteria,slection markers for both and an MCS
describe how we can use transcription factors to analyse whether 2 proteins interact
2 plasmids are present:
1. DNA binding domain with a protein of interest
2. Activation domain is with a potential protein the protein of interest interacts with
- both plamids transcribe there genes = protein of interest with DBD + potential interacting protein + activation domain
- DNA binding domain binds to promoter to promoter of reporter gene
= if potential interactor protein is asociated witb this protein it will bind AND BRING ACTIVATING DOMAIN WITH IT
- full transcription factor formed = recruits polymeraase and reproter gene is transcribed
= LacZ is an example of a reporter gene
= B-galactosidase produced and breaks down XGAL = blue/white selection
give an example of a trasncription factor and a reporter gene used to idenify protein inetarctions/interactome
GAL4 Trancription factor = DNA binding domain and activation domain split between the 2 proteins + plasmids
LacZ reporter gene:
b-galactosidase breaks down XGAL –> blue/white slection
what i used to identify intearcting proteins
yeast two-hybrid screen = identifies a single protein-protein interaction with transcription factors
