Expression vectors
Expression vectors are vectors to put inserts in that allow production of a recombinant protein in a cell
Which cell do you want to produce protein in?
- Bacteria (E. coli)
o advantages
§ well characterized
§ cheap
o disadvantages
§ prokaryote (if you want to produce eukaryotic protein this is disadvantage)
§ protein folding
§ protein modifications
- yeast or insect
o advantages
§ eukaryotic cell, so does things like a eukaryotic cell, like protein modification and folding
o disadvantages
§ more finicky
§ not as cheap as bacteria
- plant
o advantages
§ if you are creating a plant protein this is good
o disadvantages
§ finicky
§ more expensive
§ lots of unknowns
- human
o advantages
§ if you are trying to produce human protein, this is the real thing
o disadvantages
§ expensive
§ finicky
What do you need in an expression vector?
- selectable marker
- origin of replication
- site or sites to insert DNA
- sequences that enhance expression
o promoter
§ bacterial would be a good -10 and -35 box
§ eukaryotic are more complex
o intron?
§ Sometimes (like in plants) an RNA needs an intron in order to exit nucleus and be stable
o Transcription terminator
§ Especially important in eukaryotic cells
o Sequences for efficient translation
§ In bacteria, this is a Shine-Delgarno sequence
§ In eukaryotes this is the Kozak consensus
Expression vectors in agriculture
Engineering bacterial toxin that makes plants resistant to several caterpillars
- bacterium Bacillus thuringiensis produces protein toxins that kill moths and butterflies
o like European corn borer, corn rootworm, cotton bollworm
- have made plants that express this protein by recombinant DNA technology
- Ti plasmid vector
- bacterium Agrobacterium tumefaciens naturally contains a plasmid (Ti plasmid)
- infects plant, a piece of Ti plasmid (T DNA) inserts into plant chromosomal DNA
- T-DNA has genes which are very highly expressed
- make opines – source of energy for bacterium
- Ti has been souped up
o Promoter from cauliflower mosaic virus
§ Virus that infects plant cells, has promoter that plant RNAPII recognizes very well, the best plant promoter that has been found
o Intron from corn gene
o Transcriptional terminator from a tobacco gene
- insert Bt toxin in middle of T region
- put in A. tumefaciens
- punch out a disk of plant leaf material
- infect with bacterium
- allow to grow into new plant, now with all cells expressing your protein
- transgenic plant
Engineering resistance to herbicide
- broad spectrum herbicides kill most or all plants
- these are pre-emergent herbicides – farmers put them on fields before their crops emerge, to kill weeds present then
- after the crops emerge, can’t use these any more
o can use an array of specialize herbicides that kill only “weeds”
§ usually need several
o can mechanically hoe
- if crop plant could be made resistant to broad spectrum herbicide, could use these post-emergence
o simpler
o better for farmer?
- Glyphosate (Roundup) kills all plants by inhibiting an enzyme EPSPS
- There is a bacterial version of this enzyme that is not inhibited by glyphosate
- Monsanto has put this bacterial enzyme into corn, soybeans, cotton, canola
Cool expression vectors
Problems that interest me……
19, 21 (we’ve only talked about one way to do what this asks)