20 Flashcards
Key steps in producing a recombinant protein (brackets are yappage) - JELLY FISH EXAMPLE
- Isolate gene of interest (isolate mRNA and make cDNA (mature GFP) using reverse transciptiase)
- Clone into expression plasmid (need restiction sites that are complementary between plasmid vector and the gene we wanna put in, plasmid has R1 sites available, need R1 sites either side of our GFP - using PCR, make primers that are complementary to GFP (amplifying GFP), but have R1 sites off each end, then have complementary sticky ends used to paste GFP into vectors - DNA ligase)
- Transform into bacteria for expression or isolation of more DNA for use in another expression system (uses chemical transformation or elceltroporatoin) (bacteria amplyifys the plasmid - lots of copies using the Origin or replication and DNA polymerase )
- Grow cells expressing a protein of interest (antibiotic plate - only bacteria that will grow is the bacteria with plasmid with specific gene)( then put into liquid flask for more growing of bacteria containing plasmid
- Isolate and purify the protein
Key steps in procuring a recominnt protein
- Isolate gene of interest
- Clone into expression plasmid - which one?
- Transform (chemical/electricalporation) into bacteria for expression or isolation of more DNA for use in another expression system
- Growing cells expressing protein of interest
- Isolate and purify the protein
Recombinant insulin production - producing cDNA
yappage
C- chain holds A and B chain together, after a single polypeptide is produced the C chain is then cleaved out
- DNA is transcribed into mRNA after splicing to remove the introns - mRNA is then reverse transcribed into cDNA that is then cloned into plasmid
-cDNA contains its swignal, b chain, c chain and a chain
Problem with insulin being produced in bacteria and solution
- insulin is produced in the pancreas as a pre-proprotein that is further processed by golgi
- c chain is cleaved by proteases in normal eukaryotic cells but bacteria doesn’t have proteases the there is no way to produce mature insulin
- instead we produce the A and B chains separately in two different bacterial
- instead of full length cDNA we pcr amplify a cDNA copy that just contains the b chain or the a chain
- these can now be transformed to bacteria
- then purify a and b
- then mix back together in a one to one ratio under conditions that allow disulfide bonds to be formed again
Signal sequence in insulin is required for
Secreition
In a mature protein the a and b chains are held together by…
Disulphides bonds with intervening C -chains - chain must be removed
Purpose of c chain
Ensure equal amounts of a and b chain
Clone gene into expression plasmids and transform bacteria Arising problem with inserting two individual chains into bacteria and solution
When taking A and B chain and expressing them seperately in bacteria and they tried to purify the a and b chains they got a big insoluble mess as bacteria wasn’t able to fold chains properly.
Solution: tag insulin protein (subunit) of interest with a gene that produces a bacterial soluble protein (lac Z gene) thus helping solubise the insulin as well as the purification of subunits
We now have a bacterial promoter and a fusion protein of lac Z and insulin
- it is now transformed into ecology and we then purify that soluble fusion protein form the ecoli
- can now be transformed at put on antibiotic plate - select out single colonies and grow them up into a liquid culture
- but the purification of this will only get out enough for one injection, not sufficent
- pharmaceutical company uses large vats to purify it out a and b subunits from the bacteria
- everyday siphon off some of the bacteria and replace the bacteria with new stock and new media and start again
Exaltation and purification process of insulin
- Extract and purify LAC Z / insulin fusion proteins
- Treat with cyanogen bromide to cleave A and B chains
- Purify, mix A and B chains to form functional insulin (using promatography steps, separation by molecular weight or charge, depending on protein)
- then put in an environment that allows disulfide bonds to reform - exact same structure as if it was produced in ur own body - is also human so dont have complications of cow or pig insulin
Advantages of prokaryotic systems
- relatively low cost
- high yield
- pathogen fee
Disadvantages of prokaryotic systems
- proteins often partially folded
- inability to perform post-translational modifications
Why would you make recombinant insulin in ammmalian cells and why wouldn’t you
- protein can be produces as a pre-pro-protein and processed efficiently
- will be secreted from cells
- easier purification
- but more expensive to produce
How to make recominnt insulin in eukaryotic cells - what’s different
Step 1: isolate cDNA for insulin (full length - signalBCA cDNA) - amplify by restiction enzymes by PCR
Step 2: clone into eukaryotic expression plasmid
Step 3: transform bacteria to produce more plasmid DNA (bacteria wont express becuase our promoter is mammalian - bacteria is just used as a way of generating more plasmid) and then transfect eukaryotic cells
Step 4: exract recombinant insulin from cell media
Step 5: purify insulin (using chromatography)
How to decide what system to use
Therapeutic proteins - recombinant human proteins - what does it mean and what does it require
- some proteins are only active when post-trasniflionally modified
- glycosylation - requires mammalian cells e.g erythropoietin = EPO
Expression vector for EPO
Promoter - for transcription in mammalian cells (as we want to produce it in the Chinese hamster ovary cells)
- obtain human kidney RNA and reverse tracribe into cDNA - PCR with restriction sites on the end and then cut and paste it into the plasmid
- transformed into bacteria to make lots of copies
- after purifying it is then transfected into CHO cells, use big vast systems to grow them up in large amounts
- protein is secreted
- purified using chromatography
Details about AT
- AT deficiency may be hereditary (HD) or acquired
- frequency of 1 in 2000 - 5000
- increased risk of inappropriate blood clotting
- deficiency individuals receive AT when undergoing surgery or giving birth)
- AT protein expressed in the milk of transgenic goats at lactation
- AT then purified from other milk proteins
