# US 5,705,362 A · Modified signal sequences > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 5,705,362 A - Document ID: US5705362A - Status: Granted - Kind: A - Application number: 07/960,510 - Filed: 1992-10-13 - Granted: 1998-01-06 - Earliest claimed: 1992-05-25 - Assignees: Gist-Brocades N.V. - Inventors: AlfonsusJohannes Bonekamp; Marcellis Wilhelmus Elisabeth Maria Van Tilborg - Web page: https://patentgrep.com/patent/US5705362A ## CPC - C12N 9/2417: {from microbiological source} - C07K 14/485: Epidermal growth factor [EGF], i.e. urogastrone - C07K 14/5403: {IL-3} - C12N 15/625: {containing a sequence coding for a signal sequence} - C12N 15/75: for Bacillus - C07K 2319/00: Fusion polypeptide - C07K 2319/02: containing a signal sequence - C07K 2319/036: targeting to the medium outside of the cell, e.g. type III secretion - C07K 2319/75: containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - 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) ## Abstract The present invention describes a method for reducing processing ambiguity of signal peptidases. Specifically, the C-terminus of a signal sequence is mutated in a such a manner that the secreted protein contains the correct N-terminal amino acid. Furthermore, the present invention provides an hIL-3 which is essentially homogenous at its N-terminus. ## Claims ### Claim 1 A nucleic acid molecule that comprises a nucleotide sequence encoding a fusion protein of the formula signal peptide-mature protein wherein said signal peptide has an amino acid sequence modified from that of a corresponding wild-type signal peptide which contains Ala at positions -1 to -4 so that the codon at position -4 has been modified to encode Ser or Gly whereby a signal peptidase cleavage site is removed by said modification. ### Claim 2 (depends on 1) The nucleic acid molecule of claim 1 wherein the codon at position -2 has been modified to encode Ser. ### Claim 3 (depends on 1) The nucleic acid molecule of claim 1 wherein said modification is solely at position -4 of said signal peptide. ### Claim 4 (depends on 2) The nucleic acid molecule of claim 2 wherein the sequence at positions -4 to -1 of the signal peptide is selected from the group consisting of: Ser-Ala-Ser-Ala; and Gly-Ala-Ala-Ala. ### Claim 5 (depends on 1) The nucleic acid molecule of claim 1 which further comprises control sequences operably linked to said encoding nucleotide sequence capable of effecting its expression. ### Claim 6 (depends on 1) The nucleic acid molecule of claim 1 wherein the codon at position -4 has been modified to encode Ser. ### Claim 7 A recombinant host cell comprising an expression system for a nucleotide sequence encoding a precursor protein of the formula: signal peptide-mature protein wherein the signal peptide-encoding nucleotide sequence has been modified from a wild-type signal peptide which contains Ala at positions -1 to -4 so that the codon at position -4 has been modified to encode Ser or Gly so as to reduce amino terminal heterogeneity in said mature protein produced and secreted by said cells. ### Claim 8 (depends on 7) The cell of claim 7 wherein said signal peptide is the .alpha.-amylase signal peptide of a Bacillus. ### Claim 9 (depends on 7) The cell of claim 7 wherein the codon at position -2 of said signal peptide has been modified to encode Ser. ### Claim 10 (depends on 7) The cell of claim 7 wherein the codons of said nucleotide sequence at positions -4 to -1 of the modified signal peptide encode an amino acid sequence selected from the group consisting of: Ser-Ala-Ser-Ala; Gly-Ala-Ala-Ala; and Ser-Ala-Ala-Ala. ### Claim 11 (depends on 7) The cell of claim 7 wherein said codon at position -4 has been modified to encode Ser. ### Claim 12 (depends on 7) The cell of claim 7 wherein said modification is solely at position -4 of said signal peptide. ### Claim 13 (depends on 7) A method to produce a mature protein homogeneous at the N-terminus which method comprises culturing the cells of claim 7 under conditions wherein said nucleotide sequence is expressed and said mature protein is secreted; and recovering the mature protein from the culture medium. ### Claim 14 (depends on 13) The method of claim 13 wherein said mature protein is human IL-3. ### Claim 15 (depends on 13) The method of claim 13 wherein the codon at position -2 of said signal peptide has been modified to encode Ser. ### Claim 16 (depends on 13) The method of claim 13 wherein the codons of said nucleotide sequence at positions -4 to -1 of the modified signal peptide encode an amino acid sequence selected from the group consisting of: Ser-Ala-Ala-Ala; Gly-Ala-Ala-Ala; and Ser-Ala-Ser-Ala. ### Claim 17 (depends on 13) The method of claim 13 wherein the modification of said signal peptide is solely at the position -4 of said signal peptide. ### Claim 18 (depends on 13) The method of claim 13 wherein said codon at position -4 has been modified to encode Ser.