Drug Discovery 7- gene to assay Flashcards
how do we produce the protein ?
using the central dogma
- recombinant systems make use of the replication, transcription and translation machinery inside the expression host to produce a protein
- we just have to supply the cell with the right DNA and the right signals it needs to tell the cell what to do with that DNA
what is the first thing we need to assay our protein for drug binding ?
first of all we need our protein- the drug target
we also need to assay to assess efficacy - tissue based approaches are expensive and/or low throughput so generally the target needs to be expressed in a recombinant system
what does DNA look like ?
it is double stranded with the strands running in opposite directions to each other
- 2 hydrogen bonds between A and T and 3 hydrogen bonds between G and C
where is our DNA stored ?
within our chromosomes which are complex structures made up of DNA and proteins
all our cells contain the same chromosomal DNA
if all our cells contain the same chromosomal DNA why do they not all express the same genes?
only certain distinct regions of DNA are transcribed, different cell types transcribe a unique compliment of genes and they express a unique compliment of proteins
what does transcription require ?
it requires RNA polymerase to synthesise mRNA from the DNA template
- it is directed to do this by a promoter that is upstream of the gene and can be recognised by RNA polymerase
what are introns?
non- coding regions of DNA
they are removed during post translational modification by a process called splicing
other than splicing what is another process that occurs in post translational modification ?
the addition of a polyA tail to the mature mRNA
how do we obtain our protein for drug binding ?
obtain our protein by first finding the DNA encoding it and this this is cloned by isolate the mRNA encoding it
what is cDNA ?
complimentary DNA
DNA made in vitro from the mRNA isolated from cells expressing our target protein
why is cDNA different from the original genomic DNA ?
it doesnt contain any introns
how is cDNA made ?
made from mRNA using the enzyme reverse transcriptase which is derived from retrovirus
what is cloning ?
it is the process of obtaining the correct cDNA
if cells contain the same DNA why are they different ?
because it depends on the proteins in which they express which are needed for their function
what reflects the proteins that are expressed by a cell?
the mRNA content instead of the genomic DNA
what are the stages in cDNA isolation ?
1- start with tissue that expresses the protein of interest
2- extract and purify all the mRNA
3- make a cDNA library convert all the mRNA to DNA using the enzyme reverse transcriptase - this complementary DNA is called cDNA
4- isolate the cDNA encoding our protein of interest from the library using gene cloning
What does reverse transcriptase do ?
it is derived from retroviruses which use it to convert their RNA into double stranded DNA prior to integration - we use the enzyme as a tool
once the appropriate cDNA has been extracted what happens to it ?
cDNA is put into a vector so it can be expressed in a cell
- it is a means of getting the cDNA into the host cell and provides enough info to cause it to be expressed
- the vector contains a promoter which is a bit of DNA that causes RNA polymerase to transcribe the cDNA
why are strong promotors put into the vector ?
because it will lead to more copies of mRNA being produced
- these promotors are often viral e.g CMV - they are normally viral because viruses have evolved to take over a cells expression machinery and so have very strong promotors
what are the very basics required for expressing recombinant proteins ?
choose an appropriate cell line e.g. HEK293- one that will conviently express the protein and ideally one that doesnt already express the protein
choose compatible expression vector-one that recognises human RNA polymerase
inserting cDNA into vector
put this construct into the cell line= transfections
assay
where is the cDNA inserted into the vector ?
it is inserted inbetween the promotor region and the polyA sequence in the multiple cloning site
how is the cDNA inserted into the multiple cloning site ?
restriction endonucleases cut at sites in the MCS which have specific sequences that are unique to this site - they are not present elsewhere in the vector
what do the restriction endonucleases do ?
one or more restriction endonucleases is used to cut the DNA plasmid in the mutliple cloning site - breaks the covalent bonds of both strands
cDNA is then ligated into the plasmid by ligation
what are restriction endonucleases produced by ?
bacteria- named after the parent organism
how long roughly are the strands of DNA which the restriction endonuclease cuts and what is characteristic of the DNA sequence being cut?
about 4-8bp long
the DNA sequences are palindromic
what are the 2 different cleavage process that can occur when restriction endonucleases cut DNA ?
cleavage at opposite sides of the axis of symmetry produces sticky ends
cleavage at the axis generates blunt ends
what is produced when restriction enzymes cut DNA ?
there is a phosphate group at the 5’ end and an -OH group at the 3’ end
what did restriction enzymes require for activity ?
magnesium 2+ ions
what bond do restriction enzymes cleave ?
phosphodiester bond
give examples of restriction enzymes and their palindromic sequences
EcoR1- GAATTC
Pst1- CTGCAG
EcoRV- GATATC
Dpn1 - GATC
What do palindromic sequences demonstrate ?
symmetry
