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US 2002/0164621 A1Published application
Expression cloning using a tagged cDNA library
AssigneeChildren's Medical Center Corporation
InventorsLeonard I. Zon, Sadhana Agarwal
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Abstract
Methods of expression cloning where a cDNA construct expresses a tagged polypeptide for a biochemical activity of interest are described.
Claims12
A method of identifying a EDNA construct wherein the cDNA construct expresses a tagged polypeptide having a biochemical activity of interest comprising the steps of:
- a) preparing a tagged cDNA expression library comprising bacterial cells comprising tagged cDNA plasmid constructs;
- b) culturing the bacterial cells of step a) to produce clones wherein each clone corresponds to a single tagged cDNA construct;
- c) arraying the individual bacterial clones;
- d) pooling a predetermined number of arrayed clones and isolating plasmid DNA from them;
- e) transfecting suitable mammalian host cells with the pooled plasmid clones and maintaining the transfected cells under conditions suitable for the expression of the tagged cDNA construct, thereby producing tagged polypeptides;
- f) assaying the expressed tagged polypeptides for a biochemical activity of interest;
- and
- identifying a pool of clones comprising a cDNA construct encoding the tagged polypeptide having the biochemical activity of interest.
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Dependencies are inferred from the claim text.