Recombinant Proteins Flashcards
Key steps in producing recombinant protein
- Isolate gene of interest - isolate mRNA using reverse transcriptase to make cDNA without any introns
- Clone into expression plasmid - need restriction enzymes, make primers complementary that have sites which are used as sticky ends to paste our gene into the vector plasmid
- Transform into bacteria for expression or isolation of more DNA for use in another expression system - using chemical transformation or electroporation, bacteria amplifies plasmid using origin of replication and DNA polymerase
- Grow cells expressing protein of interest, bacteria plated onto antibiotic plate
- Isolate and purify the protein
Step 1 in recombinant insulin production
Isolate insulin, DNA has an intron and a C chain holding A and B chain together. DNA transcribed into mRNA and spliced to get rid of intron and cDNA made
Step 2-3 in recombinant insulin production
Protein of interest is tagged with a gene that produces a protein that is soluble
Bacterial protein that helps solubilise the insulin
A and B subunit fused to lac Z gene which helps in the purification of bacteria
Fusion protein transformed into E. coli and accumulates in cell
Step 4 in recombinant insulin production
Grow bacteria expressing insulin A and B chains
Purify out A and B subunit from bacteria
Step 5 in recombinant insulin production
Extract and purify lac Z/insulin fusion protein, treat with cyanogen bromide to cleave A and B chains, purify, mix A and B chains to form functional insulin. Separated based on molecular weight or charge
Equal amounts of A and B taken and in correct conditions, disulphide bonds form again and active insulin is made
How is insulin different?
C chain intervenes between A and B chain, to produce a mature chain we need to remove the C chain as well as disulphide bonds to be formed. Cleaved by proteases in regular cells however in bacteria does not have proteases so no way to mature insulin
Solution is to express the A and B chain separately in bacteria hence we have a cDNA copy that just contains the A chain or the B chain which are transformed, purified and mixed back together in a 1:1 ratio under conditions allowing disulphide bonds to form again
Advantages and disadvantages of prokaryotic systems
Advantages : relatively low cost, very high yield, pathogen free unlike systems used previously
Disadvantages : proteins often partially folded as not in an endogenous state, inability to perform post-translational modifications
Making recombinant insulin in mammalian cells
We can make insulin in prokaryote, mammalian and eukaryote cells. In mammalian cells, protein can be produced as a pre-pro-protein and processed efficiently, will be secreted from cells hence easier purification from media however more expensive produce
Making recombinant insulin in eukaryotic cells
Isolate cDNA for insulin- Signal BCA cDNA
Amplify with appropriate restrictions enzymes via PCR
Clone into plasmid and transform into bacteria to produce more plasmid
Transfect eukaryotic cells (introducing DNA into eukaryotic cells)
Extract recombinant insulin from cell media to make lots of insulin
Purified insulin using chromatography
What are recombinant human proteins?
Some proteins are only active when post-translationally modified. Glycosylation - requires mammalian cells e.g. erythropoietin (EPO)
What is EPO?
EPO increases RBC count leading to an increase in the oxygenation of muscle, muscle uses oxygen to burn sugar and fats to generate ATP which is required for muscle contraction
EPO is a protein that is highly post translationally modified by glycosylation which is important for biological function
Why use EPO?
Many disease states result in lowered RBC counts. Chronic renal failure can cause a decrease in EPO levels, leading to anaemia (decrease in RBC), cancer treatments (chemo) may lead to anaemia, administration of recombinant human EPO can restore RBC levels
Expression vector for EPO
Promoter that drives transcription in mammalian cells -> need to find source of EPO -> kidney mRNA reversed transcribed into cDNA to remove introns -> use PCR with restriction sites on end -> cut and paste into plasmid using restriction enzyme -> antibiotic resistance gene replicates EPO in bacteria -> transfect ovary cells -> EPO secreted out of liver to generate more blood cells -> purified using chromatography
Why would we use whole animals to make recombinant proteins?
Cells in culture cannot perform all post translational modifications equally well e.g. y-carboxylation of glutamate. Y-carboxylation of certain glutamate residues is a feature of many proteins involved in blood clotting. Imbalance in clotting can lead to excessive clotting and bleeding out
How is AT used to make recombinant proteins?
AT deficiency may be hereditary or acquired, frequency of 1 in 2000-5000. Increased risk of inappropriate blood clotting, deficient individuals receive AT when undergoing surgery or giving birth. AT protein expressed in milk of transgenic goats at lactation -> purified from other milk proteins
Human AT gene cDNA, promoter is milk specific responding to hormonal signals that induce lactation, when goat lactates it produces human AT in its milk
