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. 1978 Nov;75(11):5319–5323. doi: 10.1073/pnas.75.11.5319

Chimeric toxins: Toxic, disulfide-linked conjugate of concanavalin A with fragment A from diphtheria toxin

D Gray Gilliland *, R John Collier *,†, Joan M Moehring ‡, Thomas J Moehring ‡
PMCID: PMC392954  PMID: 281682

Abstract

A disulfide-linked conjugate of concanavalin A (Con A) and fragment A from diphtheria toxin has been synthesized and shown to be toxic for HeLa (human), Chinese hamster ovary (CHO), and SV3T3 (murine) cells. The conjugate was constructed by first coupling cystamine to Con A with a carbodiimide reagent and then reacting the modified Con A with reduced fragment A under conditions promoting disulfide interchange. The desired conjugate, obtained in nearly 50% yield relative to input of fragment A, was purified by affinity chromatography on Sephacryl S-200 and NAD-Sepharose; on analysis, it gave an average of 1.4 molecules of fragment A per tetrameric Con A molecule. The conjugate proved to be about equally active in inhibiting protein synthesis in HeLa, CHO, or SV3T3 cells in culture but was inactive relative to controls in a toxin-resistant strain of CHO cells containing altered elongation factor 2, the target protein of fragment A. With toxin-sensitive strains the conjugate was 100- to 1000-fold more active than controls, including fragment A, cystaminyl-Con A, and mixtures thereof, but was 1/50th to 1/500th as toxic as diphtheria toxin itself. Similar activity relative to controls was observed after intradermal inoculations in rabbits, and intravenous injections of the conjugate were lethal for mice. The activity of the conjugate in tissue culture was inhibited by Con A or α-methylmannoside but not by galactose. This and similar conjugates should be useful in studying mechanisms of entry of biologically active proteins into cells.

Keywords: crosslinking, affinity chromatography, protein synthesis

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Selected References

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