L17 - Recombinant proteins Flashcards
(18 cards)
what is a recombinant protein
manipulated form of a protein
= coded by a gene cloned in a system
give one reason why we migt make a recombinant protein
localisation studies
= fluorescent tag to see where protein is in cell
most commonly used expression systems
Bacteria
= high expression yield + simple genetics
safe = not contaminated with potential human pathogens = gives extra layer of security
limitations of using E.coli/ prokaryptic expression system - 2 classes of problems
- expression of eukaryotic gene in a prokaryotic system
= introns are not processed by E.coli due to no splicing = pre-mature stop codons
organisms have codon/triplet bias
- inability of bacteria to process the protein
= inability for post-translational modifications or incorrect folding
solutions to bacteria not 1. expressing the eukaryotc gene correctly or 2. not proscessing the protein correclty in expression systems
- codon optimisation = constructing the eukaryotic gene for common bacterial codon bias
use of DNA without introns
2.use of specialised strains or different vector systems if proteins not proscessed corrc;y
why are protease cleavage sites important to have in expression vectors
removal of fusion tags after protein purification
where is the shine delgarno/ribosome binding site found
10 nucleotides upstream of start codon
what is IPTG in terms of expression vectors
artificial added inducer that removes repressor proteins from the Lac Operon
= allows expression of desired gene
what is the difference between a constitutive and inducible expression vector
constitutive: uncontrolled and constant expression of gene
inducible:
controlled gene expression
what is a fusion/chimeric protein
protein consisting of ATLEAST 2 domains coded by separate genes joined so that they are transcribed and translated as a SINGLE polypeptide unit
what is a tag and give an example
short peptide sequence added to protein to identify it
= detected by antibodies against tag
histidine tail - 6xHis is a common tag
difference between tag and fusion
tag is a short peptide sequence whereas fusion proteins can e entire extra proteins or domains
= GFP = exampple of fusion
name 3 aspect to consider when tagging or adding fusion protein to target protein
ensure the tag or sequence for a fusion protein does not affect the reading frame of gene of interest
if adding at 3’ end of gene stop codon must e removed to allow it to be added to mRNA transcript
if you want the native protein following purification protease cleavage site must be added
how do we purify our desired protein
affinity purification
= use of affinity collum
what is affinity purification and how do we do it
method used to isolate a specific protein from a mixture (cell lysate)
- induce expression of protein (IPTG for example)
- fix specific resin to your tag/fusion to affinity column
- add lysate to top of collumn = it should bind to beads
- change conditions and wash again with elution buffer to get your purified protein
what is an example of an elution buffer for affinity purification
a ‘free’ version of the protein that your tag/fusion protein is bound to on beads
= outcompetes and native protein washes away with the free protein
the competing protein is bound to the ‘tag’ which is removed in the next step due to protease cleavage
how do we remove the tag/fusion protein AND the bound elution buffer if needed after affinity purification
addition of protease
= cleaves at protease cleavage sites releasing the native protein from tag bound to buffer
what kind’ve experiment would we NOT want to remove the tag/fusion protein
localisation studies
= tag is sued to view where protein is in cell
