Methods of screening and expressing cis-display libraries of disulfide-rich polypeptides
AssigneeApplied Biomedical Science Institute
InventorsDuncan MCGREGOR, Gabrielle WARNER JENKINS, Vaughn SMIDER
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Abstract
The present disclosure relates to methods of producing and screening libraries of disulfide rich proteins, for instance to identify binding peptides specific for a target molecule. In some embodiments, the binding peptides comprise disulfide rich proteins. Also provided herein are methods of producing and screening libraries comprising disulfide-rich proteins and molecular chaperones. The present disclosure also relates to methods of producing or expressing soluble disulfide rich proteins, for instance using a suitable host cell. Also provided herein are compositions comprising soluble disulfide rich proteins.
Claims29
A method of producing a disulfide rich protein (DRPs) CIS display library, the method comprising expressing a plurality of DNA constructs in a prokaryotic cell-free transcription/translation environment in the presence of at least one molecular chaperone,
- wherein the at least one molecular chaperone is provided by (i) expression from a DNA sequence encoding the molecular chaperone included in the plurality of DNA constructs, or (ii) addition of one or more DNA sequences encoding the molecular chaperone or the molecular chaperone itself to the transcription/translation environment,
- wherein each DNA construct comprises:
- i. a DNA sequence encoding one of a plurality of disulfide rich protein (DRP), wherein each of the plurality of DRPs comprises a peptide of 20-70 amino acids in length having 2-12 cysteine residues capable of forming 1-6 intramolecular disulfide bonds;
- ii. a DNA sequence encoding a DNA replication initiator protein;
- and
- iii. a DNA sequence comprising a target sequence for the DNA replication,
- thereby producing a plurality of protein fusions comprising the DRP and the DNA replication initiator protein and, optionally the molecular chaperone, from the plurality of DNA constructs,
- wherein the protein fusion binds, via the DNA replication initiator protein, to the target sequence in the DNA construct from which it was transcribed, thereby linking the protein fusion to the DNA construct to produce a DRP CIS display library comprising a plurality of DNA:protein fusions.
A method of producing a disulfide rich protein (DRPs) CIS display library, the method comprising:
- (a) providing a plurality of DNA constructs each DNA construct comprising:
- i. a DNA sequence encoding one of a plurality of disulfide rich proteins (DRPs), wherein each of the plurality of DRPs comprises a peptide of 20-70 amino acids in length having 2-12 cysteine residues capable of forming 1-6 intramolecular disulfide bonds;
- ii. one or more DNA sequences encoding a molecular chaperone, wherein the one or more molecular chaperone is trxA;
- iii. a DNA sequence encoding a DNA replication initiator protein, wherein the DNA replication initiator protein is RepA or a variant thereof that retains ability to bind to a target sequence that comprises an origin of replication sequence (ori);
- and
- iv. a DNA sequence comprising the target sequence for the DNA replication initiator protein, wherein the DNA replication initiator protein can non-covalently bind to the target sequence;
- and
- (b) expressing the plurality of DNA constructs of (a) in a prokaryotic cell-free transcription/translation environment, thereby producing a plurality of protein fusions comprising the DRP, the molecular chaperone and the DNA replication initiator protein from the plurality of DNA constructs, and
- wherein the protein fusion binds, via the DNA replication initiator protein, to the target sequence in the DNA construct from which it was transcribed, thereby linking the protein fusion to the DNA construct to produce a DRP CIS display library comprising a plurality of DNA:protein fusion proteins.
Dependencies are inferred from the claim text.