Massachusetts Institute of Technology

Millennium Pharmaceuticals Limited

Millennium Biotherapeutics, Inc.

Michael Joseph Kuranda

Sean Anthony McCarthy

Christine Ellen Bulawa

Steven Bossone

The invention involves a method of identifying nucleic acid sequences encoding signal peptide-containing proteins. The method features chimeric constructs containing a KRE9 gene that lacks a signal sequence. Yeast containing chimeric KRE9 plasmid constructs that encode signal sequences are selected based on their ability to grow on media in which sucrose is the sole carbon source.

Method for identifying genes encoding signal sequences

Disclosed are methods that can be used to (1) measure the level of polysaccharide in a sample; (2) measure the ability of a compound to degrade a polysaccharide; (3) measure the ability of a compound to modulate polysaccharide synthesis; and (4) identify or distinguish a polysaccharide, and hence organism, for diagnostic purposes in clinical medicine or research. The invention stems from Applicant's discovery that polysaccharides have multiple binding sites for polysaccharide binding moieties (PBM, e.g., wheat germ agglutinin (WGA)). In each method, one PBM links the polysaccharide to a substrate, and a tagged PBM is used to detect the polysaccharide. All of these methods can be carried out rapidly and quickly in the wells of a microtiter plate, thus permitting high through-put screening of samples or test compounds.

Cell wall assay

A gene fusion construction encoding a protein of interest and the chitin binding domain of a chitinase enzyme and the encoded fusion protein, which is the protein of interest and a protein capable of binding chitin. A plasmid vector containing the gene fusion construction is also described. The vector is used to transform host cells, which produce and secrete the recombinant fusion protein. The fusion protein is isolated by binding to chitin through its chitin-binding domain. The present method uses chitin to bind the fusion protein through its chitin-binding domain, and thereby allows immobilization and/or purification of the protein of interest, using known techniques.

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Acton, MA, United States of America

Michael Joseph Kuranda