Creation of disulfides

Figure 11.6. Schematic diagram showing the assembly of IL-12 protein for antibody-based drug delivery, (a) The mature sequences of the p35 subunit of IL-12 are fused to the C-terminus of the heavy chain of a tumour-specific antibody and co-expressed with the antibody light chain and the p40 subunit of IL-12. Formation of the final immunocytokine requires the creation of disulfide bridges between the antibody chains and interactions of p35 and p40 subunits of IL-12 [119]. (b) Alternatively the IgG heavy chain and both subunits of IL-12 can be linked via flexible linkers allowing for equimolar assembly of IL-12 [120].

Another versatile approach for the creation of artificial four-helix-bundle proteins (Scheme 21) is the assembly of a-helical peptide units via selective disulfide cross-linking, which allows for an arbitrary combination, arrangement, and orientation of the helices.[127 129]... 

SCHEME 19.11 Double-differential modification of a protein by triazole and disulfide conjugations allowed the creation of a synthetic glycoprotein reporter. 

Thus, this reagent can be used to label fluorescently proteins and other biomolecules containing free sulfhydryl residues. If there are no —SH groups available, their creation can be accomplished by reduction of indigenous disulfides or through the use of various thiolation reagents (Chapter 1, Section 4.1). 

One erf the most convenient ways erf generating sulfhydryl groups is by reduction of indigenous disulfides. Many proteins contain cystine disulfides that are not critical to structure or activity. In some cases, mild reducing conditions can free one or more —SH groups for conjugation or modification purposes. The creation erf free sulf-hydryls in this manner allows for site-directed modification at a limited number of locations within the protein molecule. 

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