Methods and compositions for protein expression and purification
AssigneeLifesensors, Inc.
InventorsTauseef R. Butt, Steven D. Weeks, Hiep T. Tran, Michael P. Malakhov, Oxana A. Malakhova
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Abstract
Methods for enhancing expression levels and secretion of heterologous fusion proteins in a host cell are disclosed.
Claims18
A method for enhancing expression levels of a protein of interest in a host cell comprising i) operably linking a nucleic acid sequence encoding molecule selected from the group consisting of SUMO, RUB, HUB, APG8, APG12, URM1, and ISG15 to a nucleic acid sequence encoding said protein of interest thereby generating a construct encoding a fusion protein, ii) introducing said nucleic acid into said host cell, whereby the presence of said molecule in said fusion protein increases the expression level of said protein of interest in said host cell.
A method for generating an altered amino terminus in a protein of interest in a host cell comprising;
- a) providing a nucleic acid sequence encoding said protein;
- b) altering the N-terminal amino acid coding sequence in said nucleic acid;
- c) operably linking a SUMO molecule to said nucleic acid sequence;
- and
- d) expressing said nucleic acid in a eukaryotic cell, thereby producing said protein of interest in said cell, said eukaryotic cell expressing endogenous SUMO cleaving enzymes, said enzyme effecting cleavage of SUMO the target protein coding sequence, thereby producing a protein of interest having an altered amino terminus.
A method for producing a sumolated protein for tracking protein localization within a host cell, comprising;
- a) providing a nucleic acid sequence encoding said protein;
- b) substituting the N-terminal amino acid coding sequence in said nucleic acid for a codon which encodes proline;
- c) operably linking a SUMO molecule to said nucleic acid sequence;
- and expressing said SUMO linked protein in said host cell.
A method for enhancing secretion levels of a protein of interest from a host cell comprising i) operably linking a nucleic acid sequence encoding molecule selected from the group consisting of SUMO, RUB, HUB, URM1, and ISG15 to a nucleic acid sequence encoding said protein of interest thereby generating a construct encoding a fusion protein, ii) introducing said nucleic acid into said host cell, whereby the presence of said molecule in said fusion protein increases the secretion of said protein of interest from said host cell.
Dependencies are inferred from the claim text.