Site-specific chemical conjugation of human Fas ligand extracellular domain using trans-cyclooctene – methyltetrazine reactions

Abstract

BackgroundFas ligand plays a key role in the human immune system as a major cell death inducing protein. The extracellular domain of human Fas ligand (hFasLECD) triggers apoptosis of malignant cells, and therefore is expected to have substantial potentials in medical biotechnology. However, the current application of this protein to clinical medicine is hampered by a shortage of the benefits relative to the drawbacks including the side-effects in systemic administration. Effective procedures for the engineering of the protein by attaching useful additional functions are required to overcome the problem.ResultsA procedure for the site-specific chemical conjugation of hFasLECD with a fluorochrome and functional proteins was devised using an inverse-electron-demand Diels-Alder reaction between trans-cyclooctene group and methyltetrazine group. The conjugations in the present study were attained by using much less molar excess amounts of the compounds to be attached as compared with the conventional chemical modification reactions using maleimide derivatives in the previous study. The isolated conjugates of hFasLECD with sulfo-Cy3, avidin and rabbit IgG Fab’ domain presented the functional and the structural integrities of the attached molecules without impairing the specific binding activity toward human Fas receptor extracellular domain.ConclusionsThe present study provided a new fundamental strategy for the production of the engineered hFasLECDs with additional beneficial functions, which will lead to the developments of the improved diagnostic systems and the effective treatment methods of serious diseases by using this protein as a component of novel molecular tools.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0381-2) contains supplementary material, which is available to authorized users.

Confirmation of covalently-linked structure and cell-death inducing activity in site-specific chemical conjugates of human Fas ligand extracellular domain

“…No appreciable m/z peak was present above 40,000, suggesting the absence of multiply-conjugated molecules (Additional file 2 ). These results showed a good agreement with the data of the SDS-PAGE and the size-exclusion chromatography analysis performed previously [ 11 ]. Consequently, it was confirmed that the conjugated sample possessed a covalent bond between the subunits belonging to hFasLECD and Avi and was suggested to be mostly composed of the 1:1 adducts of each protein.…”

“…hFasLECD-Avi conjugate was newly prepared according to the procedures in the previous paper [ 11 ]. The sample contained less than 10% of the unconjugated proteins as judged by the profile of size-exclusion chromatography (Additional file 1 ).…”

“…One of the derivatives showed the cell-death inducing activity against a human colorectal cancer cell-line, HT-29 cells, after cross-linking by an antibody [ 9 ]. The production of site-specific chemical conjugates was possible either by direct modification of the Cys residue with maleimide compounds [ 10 ] or by cyclo-addition reaction between trans -cyclooctene group (TCO) and methyltetrazine group (MTZ) [ 11 ]. The latter reaction was applicable to the conjugation of not only low molecular-weight compounds but also other functional proteins with molecular-weight similar to hFasLECD, such as egg-white avidin (Avi) and rabbit F(ab′) fragment, under mild conditions.…”