Recognition Imaging of Glycosilated Proteins (2008)

Project: Research project

Project Details

Description

Recognition Imaging of Glycosilated Proteins (2008) Recognition Imaging of Glycosilated Proteins Recognition Imaging of Glycosilated Proteins Recognition Imaging of Glycosilated Proteins The atomic force microscope (AFM) has proved to be a powerful tool for studying protein aggregates, and we have applied it to studies of peptide therapeutics produced, obtaining systematic data on aggregates as a function of the treatment of the therapeutics. There is now a recognition that aggregation of protein therapeutics can enhance immunogenicity with sometimes fatal consequences. 1 The AFM also provides a direct method for assessing the binding of a threapeutic to its target at a molecular level, using a process we call recognition imaging? We have applied it to detection and quantification of glycosilation of protein therapeutics at the single molecule level. 3 Our goal here is to combine the two techniques to address the question of how receptor binding is modified in aggregates of a therapeutic. The target for this work is a therapeutic antibody to the CD137 receptor. We have carried out recognition imaging studies of the binding of the antibody to the CD 13 7 receptor by placing the antibodies on the AFM probe and the receptors onto a mica substrate. This worked, and we obtained recognition data in good agreement with the measurements of the biological efficacy of various preparations. However, the present arrangement, in which the antibody is attached to the probe, does not allow recognition studies of aggregates of antibodies. We found that no signals were obtained when the antibodies were attached to the surface and the receptor to the probe. On looking through the sequence of the receptor, it is clear that the extracellular domain contains a number of cystienes near the N terminus. Our present chemistry for binding starts with thiolation of lysines on the protein which are subsequently linked to the AFM probe with a maleimide linkage. It seems highly likely that the maleimide attacks accessible cysteines near the N-terminus of the extracellular domain of the receptor. The goal of the current proposal is to address this problem, and produce a tip-tethered CD137 receptor with which antibody therapeutics can be assayed at the single molecule level. Protein Aggregation
StatusFinished
Effective start/end date1/1/0812/31/10

Funding

  • INDUSTRY: Domestic Company: $200,242.00

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