Drug Discovery 13 Flashcards
what is baculovirus ?
it is a natural virus that infects catepillars - it is present on leaves and is encased in a polyhedral capsid made of protein
what is a good alternative to expression in yeast ?
expression in insect cells using baculovirus
what happens when the capsid of the baculovirus is ingested ?
it is degraded and the viral particles infect the insect cells
- it replicates and produces more viral particles which are released onto the leaves as the caterpillar dies
what happens in the lab with baculovirus ?
the insect cells are grown in culture
a virus is made in which the polyhedral protein is replaced by the target protein
the virus is replicated and can infect other cells but it is not packaged into caspids, instead the cell makes large quantities of the target protein
what are stages in making a recombinant virus ?
1- produce a recombinant baculovirus transfer vector
2- then the recombinant baculovirus transfer vector and the baculogold are co-transfected into insect cells
3-this enables the recombination between the baculogold(viral DNA) and the vector within the insect cells and this causes recombinant baculovirus to be produced
4- harvest recombinant virus and amplify to produce high titer stock
5- infect cells for recombinant protein expression and purification
what are the advantages of expression in insect cells ?
levels of expression are moderate to high
post translational modification often identical to mammalian systems
what are the disadvantages of expression in insect cells ?
expensive compared with bacterial expression
how was the structure of the beta adrenoreceptor determined and ow was it expessed?
determined by x ray crystallography
protein was expressed and purified by sf9 cell (insect cell) expression system
how was the highly hydrophobic beta adrenoreceptor crystallised ?
a trick was used= a foreign protein (T4 lysozyme) was fused into an intracellular loop - inserted between 2 transmembrane helices which formed a completely separate domain but enable the proteins to form contacts that led to crystal formation
what ligand helped to solve the structure of the beta 2 adrenoreceptor ?
carazolol
