Inducible Expression of Histidine-Tagged Ricin Toxin A Chain in E. coli

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 c