Patent ID: 9550997
Date: 2017-01-24
CPC Classifications: C07K,C12N,C12P

Claim:
1. A method of identifying a secretion fusion partner (SFP), said method comprising: (i) transforming a first yeast host cell with a construct comprising a heterologous promoter operably linked to a polynucleotide encoding a secreted polypeptide; (ii) determining said secreted polypeptide to be over-secreted when the secretion level of said secreted polypeptide linked to the heterologous promoter is higher than that of said secreted polypeptide linked to a natural promoter thereof; (iii) transforming a second yeast host cell with a construct comprising a first polynucleotide encoding a target polypeptide and a second polynucleotide encoding the polypeptide determined to be over-secreted in step (ii), wherein said first and second polynucleotides are in any order relative to each other and are in the same frame; (iv) culturing said second yeast host cell under conditions wherein said construct expresses a fusion polypeptide of said target polypeptide and said over-secreted polypeptide; and (v) determining whether said fusion polypeptide is secreted into the culture medium; thereby identifying whether said over-secreted polypeptide is a SFP, wherein the SFP comprises a signal peptide, a hydrophilic domain, or a signal peptide and a hydrophilic domain, and the SFP comprises an amino acid sequence selected from the group consisting of amino acids 1-84 of SEQ ID NO: 84, amino acids 1-101 of SEQ ID NO: 84, amino acids 1-135 of SEQ ID NO: 84, amino acids 1-169 of SEQ ID NO: 84, amino acids 1-195 of SEQ ID NO: 84, amino acids 1-227 of SEQ ID NO: 84, amino acids 1-271 of SEQ ID NO: 84, amino acids 1-364 of SEQ ID NO: 84; or the SFP is selected from the group consisting of BGL2 (SEQ ID NO: 80), GAS3 (SEQ ID NO: 81), GAS5 (SEQ ID NO: 82), PST1 (SEQ ID NO: 83), SCW4 (SEQ ID NO: 84), SCW10 (SEQ ID NO: 85), SIMI (SEQ ID NO: 86), UTH1 (SEQ ID NO: 87), YGP1 (SEQ ID NO: 88), YPS1 (SEQ ID NO: 89), and ZPS1 (SEQ ID NO: 90); and wherein the target polypeptide is selected from the group consisting of an interleukin, a coagulation factor, an interferon-α, -β or -γ, a granulocyte-colony stimulating factor, a granulocyte macrophage-colony stimulating factor, a tissue growth factor, an epithelial growth factor, a TGFα, a TGFβ, an epidermal growth factor, a platelet-derived growth factor, a fibroblast growth factor, a follicle stimulating hormone, a thyroid stimulating hormone, an antidiuretic hormone, a pigmentary hormone, a parathyroid hormone, a luteinizing hormone-releasing hormone, a carbohydrate-specific enzyme, a proteolytic enzyme, a lipase, an oxidoreductase, a transferase, a hydrolase, a lyase, an isomerase, a ligase, an immunoglobulin, a cytokine receptor, a lactoferrin, a phospholipase A2-activating protein, an insulin, a tumor necrosis factor, a calcitonin, a calcitonin gene related peptide, an enkephalin, a somatomedin, an erythropoietin, a hypothalamic releasing factor, a prolactin, a chorionic gonadotropin, a tissue plasminogen activator, a growth hormone releasing peptide, a thymic humoral factor, an anticancer peptide, and an antibiotic peptide.