Lecture 20: USING RECOMBINANT DNA TECHNOLOGIES TO MAKE PROTEINS Flashcards
What is the first step of producing a recombinant protein?
Isolate gene of interest
What happens after isolating the gene of interest?
Clone into expression plasmin (with correct promoter)
What happens after clones into expression plasmid?
Transform into bacteria for expression or isolation of more DNA for use in another expression system
What happens after transform into bacteria for expression or isolation of more DNA for use in another expression system?
Grow cells expressing protein of interest
What happens after grow cells expressing protein of interest?
Isolate and purify the protein
What is involved in isolating the gene of interest?
DNA (with introns)»_space; mRN (with introns)»_space;> cDNA (without introns) using reverse transcriptase
Where is insulin produced?
In the pancreas as a pre-proprotein that is further processed by Golgi which won’t happen in bacteria
What is the solution to insulin needing to be processed?
Express chain A and B in separate bacteria
What happens in bacteria 1?
Promoter, lac Z gene, insulin gene A subunit, antibiotic resistance gene = insulin A protein accumulates
What happens in bacteria 2?
Promoter, lac Z gene, insulin gene B subunit, antibiotic resistance gene = insulin B protein accumulates
What happens after the insulin A and B proteins accumulate?
Grow the bacteria expressing insulin A and B chains (selected using antibiotic)
What is the first step of isolating and purifying the protein?
Extract and purify lac Z/insulin fusion proteins
What happens after lacZ/fusion proteins are extracted and purified?
Treat with cyanogen bromide to cleave A and B chains (no lacZ)
What happens after the protein is treated with cyanogen bromide?
Purify, mix A and B chains to form functional insulin (joined by a disulfide bond)
What are the advantages of prokaryotic systems?
Relatively low cost, high yield and pathogen free
What are the disadvantages of prokaryotic systems?
Proteins are often partially folded and the inability to perform post-translational modifications
What are the advantages of recombinant insulin in mammalian cells?
Protein can be produced as pre-proprotein and processed efficiently, it will also be secreted from cells for easier purification
What is the disadvantage of recombinant insulin in mammalian cells?
More expensive to produce
What are the differences when making insulin in eukaryotic cells?
The promoter is specific to eukaryotic cells, transfected onto eukaryotic cells and there is no need to form disulphide bonds as they are already presens
When are some proteins active?
Only when post-translationally modified
What is an example of glycosylation?
Erythropoietin (requires mammalian cells)
What does increase in RBC lead to?
An increase in the oxygenation of muscle
What do muscles use?
Oxygen to burn sugar and fats to generate ATP
What is required for muscle contraction?
ATP
What do many disease states result in?
lowered RBC count
What can chronic renal failure cause?
A decrease in EPO levels, leading to anaemia (decreased RBC levels)
What can cancer treatments lead to?
Anaemia
What can administration of recombinant human EPO do?
Restore RBC levels
When was the EPO gene cloned?
In the early 1980’s
What is done with the EPO protein?
It is post-translationally modified (glycosylation - addition of carbohydrates to asparagine, serine or threonine residues)
What is glycosylation of EPO important for?
Biological fucntion
What is EPO made in?
Chinese hamster ovary (CHO) cells
What can cells in culture not do?
Perform all post-translational modifications equally well, such as gamma carboxylation of glutamate
What is gamma carboxylation of glutamate residues a feature of?
Many proteins involved in blood clotting
What happened in 2006?
The first recombinant protein transgenic animal was approved as a drug (anti-thrombin -AT)
What may AT deficiency be?
Hereditary or acquired
What is the frequency of AT deficiency?
1 in 2000-5000
What does AT deficiency increase the risk of?
Inappropriate blood clotting
When do AT deficient individuals receive AT?
when undergoing surgery or giving birth
What is AT protein expressed in?
The milk of transgenic goats at lactation then purified from other milk proteins
What does the milk specific promoter do?
Responds to hormonal signals that induce lactation
