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Improved procedure for direct coupling of carbohydrates to proteins via reductive amination

  1. Author:
    Gildersleeve, J. C.
    Oyelaran, O.
    Simpson, J. T.
    Allred, B.

  2. Author Address

    Gildersleeve, Jeffrey C.; Oyelaran, Oyindasola, Allred, Benjamin] NCI, Ctr Canc Res, Med Chem Lab, NIH, Frederick, MD 21702 USA. [Simpson, John T.] NCI, Prot Chem Lab, Adv Technol Program, SAIC Frederick,Inc, Frederick, MD 21702 USA.
    1. Year: 2008
  2. Journal: Bioconjugate Chemistry
    1. 19
    2. 7
    3. Pages: 1485-1490
  4. Type of Article: Article
  1. Abstract:

    Carbohydrate-protein conjugates are utilized extensively in basic research and as immunogens in a variety of bacterial vaccines and cancer vaccines. As a result, there have been significant efforts to develop simple and reliable methods for the construction of these conjugates. While direct coupling via reductive amination is an appealing approach, the reaction is typically very inefficient. In this paper, we report improved reaction conditions providing an approximately 500% increase in yield. In addition to optimizing a series of standard reaction parameters, we found that addition of 500 mM sodium sulfate improves the coupling efficiency. To illustrate the utility of these conditions, a series of high mannose BSA conjugates were produced and incorporated into a carbohydrate microarray. Ligand binding to ConA could be observed and apparent affinity constants (K(d)s) measured using the array were in good agreement with values reported by surface plasmon resonance. The results show that the conditions are suitable for microgram-scale reactions, are compatible with complex carbohydrates, and produce biologically active conjugates.

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External Sources

  1. PMID: 18597509

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