Method for refolding enzymes
AssigneeElan Pharmaceuticals, Inc.
InventorsAlfredo Tomasselli, Robert Heinrikson, Donna Paddock, Ana Mildner, Thomas Emmons
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Abstract
The invention provides methods for efficient recombinant expression, refolding, and purification of Beta-site APP cleaving enzyme (BACE) polypeptides. In various aspects, the method includes the steps of expressing a recombinant construct in bacteria, dissolving inclusion bodies with a denaturant at high pH in the presence of a reducing agent, diluting the solubilized BACE polypeptide in an aqueous solution at a temperature of about 1° C. to 15° C., and incubating the diluted sample at a temperature of about 4° C. to 15° C. until the recombinant BACE polypeptide folds into an active enzyme.
Claims24
A method for producing an active recombinant human Beta-site APP cleaving enzyme (BACE) polypeptide comprising:
- a) expressing a polynucleotide sequence encoding a BACE polypeptide lacking all or a portion of the sequence corresponding to residues 1-66 of SEQ ID NO:
- 1,
- b) solubilizing the BACE polypeptide in a denaturant at a pH from about 10 to about 11 and in the presence of a reducing agent;
- c) diluting the solubilized BACE polypeptide in an aqueous solution having a temperature of about 1° C. to about 15° C., to obtain a diluted sample;
- and
- d) incubating the diluted sample at a temperature of about 4° C. to about 15° C. until the BACE polypeptide folds into an active enzyme.
A method for producing an active recombinant human BACE polypeptide comprising:
- a) expressing a polynucleotide encoding the BACE polypeptide in a host cell to produce a BACE polypeptide inclusion bodies;
- b) solubilizing the BACE polypeptide inclusion bodies to release the recombinant human BACE polypeptide;
- c) reducing the released BACE polypeptide with a reducing agent;
- d) diluting the reduced BACE polypeptide with an aqueous solution having a temperature of about 1° C. to about 15° C.;
- and
- e) incubating the diluted BACE polypeptide at a temperature from about 4° C. to about 15° C. and at a pH from about 10 to about 11, to obtain the active recombinant human BACE polypeptide.
A method for refolding an active recombinant human BACE polypeptide comprising:
- a) diluting a solubilized and a reduced BACE polypeptide with a low ionic strength aqueous solution having a temperature of about 1° C. to about 15° C.;
- b) incubating the diluted BACE polypeptide at a starting pH of about 10 to about 11, and at a temperature of about 1° C. to about 15° C. for about 2 days to about 6 weeks to allow the BACE polypeptide to fold into an active enzyme;
- and
- c) recovering the active refolded BACE polypeptide.
Dependencies are inferred from the claim text.