Using Recombinant DNA Technologies to Make Proteins Flashcards
What are the key steps in producing a recombinant protein?
- Isolate gene of interest
- Close ino expression plasmid
- Transform into bacteria for expression or isolation of more DNA for use in another expression system
- Grow cells expressing protein of interest in the appropriate expression system (if bacteria are the expression system and the factory then the protein will be generated at the same time as the vectors)
- Isolate and purify the protein
Describe the process of creating recombinant insulin
- Obtain human insulin cDNA
- Insulin is complicated so have to start with Insulin gene, which makes a protein called Preproinsulin. The cells then process this into proinsulin (by removing signal peptide), and then further process it to remove the whole c-peptide which creates mature insulin.
- Insulin is produced in the pancreas as a pre-proprotein that is further processed in the Golgi. This won’t happen in bacteria so the solution is to express chain A and chain B separately in bacteria - clone gene into expression plasmids
- transform bacteria (into two separate bacterial colonies)
- Grow bacteria expressing insulin A and B chains
- Extract and purify insulin (combine the chains to produce functional insulin)
What are the advantages and disadvantages of prokaryotic systems?
Advantages:
- relatively low cost
- high yield (bc bacteria replicate so fast)
- pathogen free (because prokaryotes have no potential to contaminate protein samples with eukaryotic pathogens)
Disadvantages:
- proteins often partially folded (because of different cytoplasm environment in bacteria)
- inability to perform stable post-translational modifications (don’t do so in a reliable manner. add them on but don’t form covalent attachment properly so they just fall off again)
Why don’t we generally use mammalian cells to make recombinant insulin instead?
- Protein can be produced as a pre-pro-protein and processed efficiency
- The protein wold be secreted from cells (easier purification)
- BUT its more expensive to produce and also has much lower yield
What do we do differently when making recombinants in eukaryotic cells?
- Isolate cDNA for insulin
- Clone EUKARYOTIC expression plasmid
- Transform BACTEIRA to produce more plasmid DNA
- Transfect EUKARYOTIC cells
- Extract recombinant insulin rom cell media
- Purify insulin
What factors do you need to consider when choosing the most appropriate expression system?
- Speed (bacteria best)
- Cost (bacteria best)
- Post translational modification ability (mammalian best)
- Folding (mamallian best)
Describe erythropoietin (EPO) and what the glycosylation of rEPO does
Protein pos-translationally modified (glycosylation - addition of carbs to AA’s)
- rEPO made in Chinese hamster ovary (CHO) cells
Glycosylation of EPO:
- increases the stability in the bloodstrea
- required for proper biological function
- helps the protein solublise in the bloodstream
- ensures appropriate immune recognition
(need all four of these sites to be glycosylated for it to function properly)
- Due to post translational mod. rEPO production needs to be done in mammalian cells!
Why would we want to make recombinant EPO?
- Many disease states result in lowered red blood cell counts
- Chronic renal failure can cause a decrease in EPO levels, leading to anaemia (decrease in RBC levels)
- Cancer treatments (chemotherapy) may lead to anemia
- Administration of rEPO can restore red blood cell levels
- Need to be injected bc if taken orally the digestive system will just break down the proteins
What are the differences in Natural EPO and rEPO?
Natural EPO:
- produces primarily in the kidneys and gas a glycosylation pattern that is specific to human cells (only four glycosylations)
rEPO:
- produces in Chinese hamster ovary (CHO) cells. these cells add sugars in a manner that can differ significantly from human cells (might add more glycosylatuons or in a diff location)
How is rhEPO detected?
Detected in isoelectric focusing (IEF)
- separates proteins based on their isoelectric point
- hEPO and ehEPO have different charges die to their different glycosylation patterns
- Gel has a pH gradient. proteins stop moving in an electric field once they reach their pI
- hEPO and rhEPO will stop moving at different locations in the gel
- visualised via vining of specific anti-EPO antibodies
Describe pharming in the context of antithrombin (AT)
Using whole animals to make recombinant proteins
- Cells in culture cannot perform all post-translational modification equally well
- eg. gamma-carboxylation of glutamate
- gamma-carboxylation of certain glutamate residues is a feature of many proteins involved in blood clotting
- The first recombinant protein produced from a transgenic animal approved as a drug was antithrombin (AT)
- AT deficiency may be hereditary (AD) or aquired
- increased risk of inappropriate blood clotting
- Deficient individuals receive AT when undergoing surgery or giving birth
- AT protein expressed in the milk of transgenic goats at lactation (responds to hormonal signals that induce lactation, and has a lactation specific promotor so will only be expressed in the mammary glands)
- AT then purified from other milk proteins
Describe the process of creating cloned transgenic offspring that produce AT in their milk
- Mammary gland specific regulatory sequences + genes for antithrombin
- genes introduced into somatic cells and isolate oocytes and enucleate (remove nuclei)
- fuse transgenic cell with enucleated oocyte
- oocytes transferred into surrogate female
- cloned transgenic offspring that produce AT in their milk
