# US 11,306,127 B2 · Compositions and methods for producing high secreted yields of recombinant proteins > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 11,306,127 B2 - Document ID: US11306127B2 - Status: Granted - Kind: B2 - Application number: 15/920,291 - Filed: 2018-03-13 - Published: 2018-10-04 - Granted: 2022-04-19 - Earliest claimed: 2017-03-10 - Assignees: Bolt Threads, Inc. - Applicants: Bolt Threads, Inc. - Inventors: Joshua Kittleson; Thomas Stevens; Rena Hill; Carlos Gustavo Pesce; David N. Breslauer; Daniel M. Widmaier - Web page: https://patentgrep.com/patent/US11306127B2 ## CPC - C07K 14/43586: {from silkworms} - C07K 14/395: from Saccharomyces - C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione - C07K 2319/02: containing a signal sequence - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)} ## Abstract The present disclosure relates to methods for producing recombinant proteins, as well as compositions used in and produced by such methods. Specifically, the present disclosure relates to methods for producing high secreted yields of recombinant proteins, and the compositions provided herein include expression constructs, recombinant vectors, and recombinant host cells that comprise polynucleotide sequences encoding proteins operably linked to recombinant secretion signals that comprise the leader peptide of the α-mating factor (αMF) of Saccharomyces cerevisiae and a non-αMF signal peptide. ## Claims ### Claim 1 An expression construct comprising a polynucleotide sequence that encodes a protein operably linked to a recombinant secretion signal, wherein said recombinant secretion signal comprises a leader peptide and a signal peptide, wherein said leader peptide comprises the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein said signal peptide comprises the polypeptide sequence of SEQ ID NO: 9, and wherein said protein is a silk protein. ### Claim 2 (depends on 1) The expression construct of claim 1, wherein said leader peptide sequence consists of the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. ### Claim 3 (depends on 1) The expression construct of claim 1, wherein said signal peptide sequence consists of the polypeptide sequence of SEQ ID NO: 9. ### Claim 4 (depends on 1) The expression construct of claim 1, wherein said expression construct comprises multiple copies of said polynucleotide sequence. ### Claim 5 (depends on 1) The expression construct of claim 1, wherein said protein comprises the polypeptide sequence of SEQ ID NO: 17. ### Claim 6 (depends on 1) A recombinant vector comprising the expression construct of claim 1. ### Claim 7 A recombinant host cell comprising an expression construct comprising a polynucleotide sequence that encodes a protein operably linked to a recombinant secretion signal, wherein said recombinant secretion signal comprises a leader peptide and a signal peptide, wherein said leader peptide comprises the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, wherein said signal peptide comprises the polypeptide sequence of SEQ ID NO: 9, and wherein said protein is a silk protein. ### Claim 8 (depends on 7) The recombinant host cell of claim 7, wherein said recombinant host cell is a yeast cell. ### Claim 9 (depends on 8) The recombinant host cell of claim 8, wherein said recombinant host cell is a budding yeast cell. ### Claim 10 (depends on 8) The recombinant host cell of claim 8, wherein said recombinant host cell is a methylotrophic yeast cell. ### Claim 11 (depends on 10) The recombinant host cell of claim 10, wherein said recombinant host cell is Pichia pastoris. ### Claim 12 (depends on 7) The recombinant host cell of claim 7, wherein said expression construct is stably integrated within the genome of said recombinant host cell. ### Claim 13 (depends on 7) The recombinant host cell of claim 7, wherein said expression construct is maintained extrachromosomally in said recombinant host cell. ### Claim 14 (depends on 7) A composition comprising the recombinant host cell of claim 7 and a culture medium. ### Claim 15 (depends on 1) The expression construct of claim 1, wherein said leader peptide sequence consists of the polypeptide sequence if SEQ ID NO: 1 or SEQ ID NO: 2, and wherein said signal peptide sequence consists of th polypeptide sequence of SEQ ID NO: 9. ### Claim 16 (depends on 7) A method for producing a silk protein, comprising the steps of: a) culturing the recombinant host cell of claim 7 in a culture medium; and b) extracting said silk protein from the culture medium.