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.
Highlights
Fas ligand plays a key role in the human immune system as a major cell death inducing protein
For the preparation of hFasLECD-TCO and hFasLECD-MTZ, the reactive cysteine residue in the N-terminal tag sequence of hFasLECD molecule was chemically modified with a large excess molar amount of trans-cyclooctene-PEG3-maleimide (TCO-PEG3-MAL) and methyltetrazine-PEG4maleimide (MTZ-PEG4-MAL) reagents, respectively
The tertiary structure of a complex between hFasLECD and human decoy receptor 3 (DcR3) has been determined by X-ray crystallography, which serves as a model for hFasLECD – hFasRECD complex [26]
Summary
Fas 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 is expected to have substantial potentials in medical biotechnology. Site-specific chemical conjugation utilizing a reactive tag residue to install chemical groups by covalent additions is another potent technology for engineering proteins to attach new functionalities, which are not available in the original molecules [17, 18]. TCO and MTZ groups are fairly stable in physiological aqueous buffer solutions, and the conjugation reaction between them can proceed with exceptionally fast kinetics and high selectivity [22, 23] This makes the reaction attractive for the applications in which only a limiting amount of molecules to be conjugated are generally available, such as the cases using expensive low molecular-weight compounds or precious functional proteins. In spite of its potential usefulness, the behaviors in actual conjugation events are not always well documented yet
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