D2C7-IT (D2C7) was expressed in E. coli BL21 (λ DE3) (Agilent), under the control of T7 promoter. D2C7 from inclusion bodies was reduced, refolded, and further purified as monomers by ion exchange and size-exclusion chromatography as described previously (15 (link)). Finally, endotoxin removal from the purified immunotoxin was achieved with ActiClean Etox resin (Sterogene). The purity of the final D2C7 preparation was >95% by SDS-PAGE and size-exclusion chromatography (HPLC).
Protocol detail
Purification of Immunotoxin D2C7
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E coli
Endotoxin
Hplc
Immunotoxin
Inclusion bodies
Ion exchange
Resin
Sds page
Size exclusion chromatography
Corresponding Organization : Duke Medical Center
Other organizations : Duke University, University of Colorado Anschutz Medical Campus, Memorial Sloan Kettering Cancer Center, National Cancer Institute, National Institutes of Health, Center for Cancer Research
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Variable analysis
independent variables
- Expression of D2C7 in E. coli BL21 (λ DE3) under the control of T7 promoter
- Purity of the final D2C7 preparation (>95% by SDS-PAGE and size-exclusion chromatography (HPLC))
- Endotoxin removal from the purified immunotoxin (achieved with ActiClean Etox resin)
- Reduction, refolding, and purification of D2C7 from inclusion bodies as described previously (15)
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
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