Enzymatic synthesis of antibody-human serum albumin conjugate for targeted drug delivery using tyrosinase from Agaricus bisporus
2012; Royal Society of Chemistry; Volume: 3; Issue: 5 Linguagem: Inglês
10.1039/c2ra22560c
ISSN2046-2069
AutoresAlexandra Rollett, Barbara Thallinger, Anna Ohradanova‐Repic, Christian Machacek, Evelyn Walenta, Artur Cavaco‐Paulo, Ruth Birner‐Gruenberger, Juliane Gertrude Bogner‐Strauß, Hannes Stockinger, Georg M. Guebitz,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoHighly specific targeted drug delivery devices can be obtained with antibody-human serum albumin (mAb-HSA) conjugates. However, their conventional production involves several reaction steps including chemical modification and activation of both proteins followed by cross-linking often involving toxic chemicals. Here, we describe the enzymatic synthesis of mAb-HSA conjugates for targeted drug delivery devices using tyrosinase from Agaricus bisporus under mild reaction conditions (pH 6.8, 25 °C). Reaction conditions were optimized by using fluorescence labeled HSA to facilitate SDS-PAGE analysis with fluorescence scanning. Enzymatic cross-linking in the presence of natural low molecular weight phenolic compounds (e.g. caffeic acid) resulted in reaction products in the molecular weight range of ∼216 kDa, corresponding to mAb-HSA conjugates. The composition of the conjugates was confirmed with tryptic digestion followed by LC-MS/MS analysis of the resulting peptide fragments. Successful binding of mAb-HSA conjugates (in contrast to free HSA) to MHC II molecules, located on antigen-presenting cells, was demonstrated by both ELISA and flow cytometry analysis.
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