Validation of a mutant of the pore-forming toxin sticholysin-I for the construction of proteinase-activated immunotoxins.

The use of pore-forming toxins from sea anemones (actinoporins) in the construction of immunotoxins (ITs) against tumour cells is an alternative for cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs.

"Reverse-staining" of biomolecules in electrophoresis gels: analytical and micropreparative applications.

Negative or reverse staining using imidazole and zinc salts for protein detection in electrophoresis gels was originally introduced in 1990. The method is based on the selective precipitation of zinc imidazolate in the gel except in the zones where proteins are located. The method was later adapted to allow high-sensitivity negative detection of nucleic acids and bacterial lipopolysaccharides. It provides a practically quantitative recovery of intact biomolecules and is a method of choice for micropreparative applications of gel electrophoresis to proteomics and similar structural studies. Zinc-mediated protein fixation in the gel is fully reversible and the eluted biomolecules are neither chemically modified nor contaminated with organic dyes. Here we present a detailed compilation of practical methods for implementing these techniques with emphasis in their analytical or micropreparative applications.