This article details a protocol for expressing the ricin toxin A chain (RTA) in E. coli using a lac operator-regulated T7 polymerase system. The method involves transforming E. coli with a plasmid that carries the RTA gene and an antibiotic resistance gene, allowing for selective growth and protein expression.
Begin with E. coli colonies containing a lac operator-regulated T7 polymerase gene and an expression plasmid.
The plasmid carries genes for antibiotic resistance and the ricin toxin A chain.
The resistance gene is constitutively expressed, producing an antibiotic resistance protein.
The RTA gene is histidine-tagged and regulated by a T7 promoter and lac operator.
Inoculate the colonies into antibiotic-supplemented media and incubate with shaking.
The antibiotic resistance protein inactivates the antibiotic, enabling selective growth of plasmid-bearing bacteria.
Transfer the culture to antibiotic-supplemented media and incubate with shaking to promote bacterial growth.
Lower the culture temperature to enhance proper folding of the RTA protein.
Add an inducer to relieve lac repression. This triggers T7 polymerase expression, initiating RTA transcription and protein synthesis.
Centrifuge to collect the bacteria and discard the supernatant.
Wash with a buffer, centrifuge, and remove the supernatant.
Resuspend the His-tagged RTA-expressing bacteria in buffer.
Begin this protocol with cell transformation as detailed in the text protocol. Inoculate five milliliters of LB kanamycin medium with cells containing RTA expression plasmid or the empty vector.
Incubate the cells at 37 degrees Celsius and 220 RPM for 24 hours. Supplement one liter of LB kanamycin medium with five milliliters of preculture. Then incubate the cells at 37 degrees Celsius and 220 RPM until cells have reached an optical density at 600 nanometers of 0.8 to 1.0.
Thereafter, reduce the culture temperature to 28 degrees Celsius. Induce RTA expression of the E. Coli by adding IPTG to a final concentration of one millimolar. After 3.5 hours at 28 degrees Celsius and 220 RPM, harvest the cells by centrifugation at 10,000 g and four degrees Celsius for 10 minutes.
Then, wash the pellet with five milliliters of binding buffer and pellet at 10,000 g and four degrees Celsius for 10 minutes. After repeating the wash once, resuspend the pellet in five milliliters of binding buffer.
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