Solubility reporter gene constructs

A method of identifying mutations in a cell that alter the solubility of a target polypeptide comprising:

• a) treating a cell with a mutagen;
• b) introducing into the cell:
• i) a solubility reporter nucleic acid that comprises a protein solubility responsive promoter operably linked to a reporter gene;
• and
• ii) a target polypeptide-expressing nucleic acid that comprises a polynucleotide that encodes a target polypeptide;
• c) culturing the cell under conditions favorable for expression of the target polypeptide;
• d) measuring expression of the reporter gene;
• and
• e) comparing the level of expression of the reporter gene in the cell with the level observed in an unmutated cell that comprises the solubility reporter nucleic acid and the target polypeptide-expressing nucleic acid to identify a cell that comprises a mutation that alters the solubility of the target polypeptide.

A method for identifying alterations to a polynucleotide that encodes a target polypeptide that alter the solubility of the target polypeptide, the method comprising:

• a) altering a polynucleotide that encodes the target polypeptide to form an altered polynucleotide;
• b) introducing into a cell:
• i) a solubility reporter nucleic acid that comprises a protein solubility responsive promoter operably linked to a reporter gene;
• and
• ii) a target polypeptide-expressing nucleic acid that comprises the altered polynucleotide;
• c) culturing the cell under conditions favorable for expression of the target polypeptide;
• d) measuring the expression of the reporter gene;
• and
• e) comparing the level of expression of the reporter gene with the level observed in a cell with an unaltered polynucleotide that encodes the target polypeptide, to identify an alteration to the polynucleotide that changes the solubility of the encoded target polypeptide.

A method to identify variations in a process for biosynthesis of a target polypeptide that alter the solubility of the target polypeptide, the method comprising:

• culturing a host cell under alternative conditions in which the target polypeptide is expressed, wherein the host cell comprises:
• a) a solubility reporter nucleic acid that comprises a protein solubility responsive promoter operably linked to a reporter gene;
• and
• b) a target polypeptide-expressing nucleic acid that comprises a polynucleotide that encodes a target polypeptide;;
• and
• comparing the expression of the reporter gene by host cells grown under each of the alternative conditions.

A method of screening an expression library to identify library members that express soluble target polypeptide, the method comprising:

• a) introducing a plurality of expression vectors that each comprise a polynucleotide that encodes a target polypeptide into a plurality of host cells to create an expression library, wherein the host cells comprise a solubility reporter nucleic acid that comprises a protein solubility responsive promoter operably linked to a reporter gene;
• b) culturing the host cells under conditions in which the target polypeptides are expressed;
• and
• c) detecting expression of the reporter gene, thereby identifying library members that express soluble target polypeptides.

A method of identifying a promoter that is differentially regulated in response to expression of an insoluble polypeptide in a host cell that comprises the promoter, the method comprising:

• a) providing a host cell that comprises:
• i) a solubility reporter nucleic acid that comprises a putative protein solubility responsive promoter operably linked to a reporter gene;
• and
• ii) a target polypeptide-expressing nucleic acid that comprises a polynucleotide that encodes a target polypeptide;
• b) culturing the host cell under conditions in which the target polypeptide is expressed in insoluble form;
• and
• c) determining whether expression of the reporter gene is increased or decreased, thereby determining whether the putative protein solubility responsive promoter is differentially regulated in response to expression of an insoluble polypeptide in the host cell.