Lecture 9 - Uses of Recombinant DNA: Damian Parry Flashcards
What are the 3 uses of recombinant DNA once we have obtained it?
- Induce expression of protein in a host.
- Recover plasmid and manipulate further.
- Recover plasmid and transfer to another cell type.
What are the desired properties of an expression vector?
Must contain a promoter sequence that is recognized by the host cell’s RNA polymerase.
Gene to be expressed must be ligated at the 3’ end of the promoter.
Define intron
A nucleotide sequence within a gene that is removed by RNA splicing.
Define Exon
A part of a gene that will encode a part of the final mature RNA after introns have been removed by splicing.
Explain how reverse transcriptase turns RNA into cDNA
Reverse transcriptase starts at the 3’ end of mRNA. A 5’ primer then binds to the transcriptase. The transcriptase then slides up the mRNA towards the 5’ end, and in doing so creates the cDNA strand. The mRNA then detatches leaving a single stranded cDNA with no introns. DNA polymerase then can be used to create a x2 stranded cDNA.
What are the advantages of bacterial expression systems?
4
Simple cells
Short generation time
and large yield
Low Cost
Few ethical issues
What are the disadvantages of bacterial expression systems?
4
Eukaryotic proteins can fail to fold correctly and lose biological activity.
Proteins can be toxic to the bacterial cell.
No post-translational modifications e.g addition of phosphates and sugars.
Cell wall can be difficult to work with.
What are the advantages of Yeast expression Systems?
4
Simple unicellular eukaryote
Resembles mammalian cells
Grows quick and is cheap
Performs post-translational modifications.
What are the disadvantages of Yeast expression systems?
2
Contains proteases which can degrade some recombinant proteins.
Post translational modifications can differ from mammalian cells.q
What are the advantages of Insect Cell expression systems?
4
High level protein expression
Correct folding of mammalian proteins
Post translational modifications
Cheaper than mammal cell culture.
What is the disadvantage of using an insect cell expression system?
(1)
Post translational modifications may differ from mammalian cells.
What is the advantage of using mammalian expression systems?
2
Easily produces and correctly folds mammalian proteins (obviously).
Post translational modifications.
What are the disadvantages of using mammalian expression systems?
(3)
Complex cells
Low cell density
Expensive
What are the issues in trying to obtain insulin from animals?
They are not identical - so potential side effects
Difficult to purify
Possible contamination with pathogens such as BSE.
What is the basic structure of insulin?
2 polypeptide chains joined by disulphide bonds (see Stryer p.36)
