Artificial secretion peptides for heterologous protein production

Abstract

Provided herein, in some embodiments, are artificial secretion peptides capable of directing secretion from Lactobacillus for use, for example, in producing heterologous proteins, including therapeutic proteins.

What is claimed is: 
     
         1 . An artificial secretion signal peptide comprising an amino acid sequence that has at least 90% identity to an amino acid sequence of any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 11, and SEQ ID NO:7. 
     
     
         2 . The artificial secretion signal peptide of  claim 1 , wherein the amino acid sequence has at least 95% identity to an amino acid sequence of any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 11, and SEQ ID NO:7. 
     
     
         3 . The artificial secretion signal peptide of  claim 2 , wherein the amino acid sequence has at least 98% identity to an amino acid sequence of any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 11, and SEQ ID NO:7. 
     
     
         4 . The artificial secretion signal peptide of  claim 3 , wherein the amino acid sequence comprises the amino acid sequence of any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO: 11, and SEQ ID NO:7. 
     
     
         5 . A protein fused to the artificial secretion signal peptide of any one of  claims 1 - 4 . 
     
     
         6 . The protein of  claim 5 , wherein the artificial secretion signal peptide is fused to the N-terminus of the protein. 
     
     
         7 . The protein of  claim 5  or  6 , wherein the protein is a therapeutic protein. 
     
     
         8 . The protein of  claim 7 , wherein the protein is an antibody, optionally wherein the antibody binds specifically to a viral antigen or a microbial antigen. 
     
     
         9 . The protein of  claim 7 , wherein the therapeutic protein is a cytokine, optionally wherein the cytokine is IL-10. 
     
     
         10 . The protein of  claim 7 , wherein the therapeutic protein is an endocrine hormone, optionally wherein the endocrine hormone is glucagon-like peptide 1 (GLP1). 
     
     
         11 . The protein of  claim 7 , wherein the therapeutic protein is an antimicrobial peptide, optionally wherein the antimicrobial peptide is human beta defensin 1 (hBD1). 
     
     
         12 . A nucleic acid encoding the artificial secretion signal peptide of any one of  claims 1 - 4  or the protein of any one of  claims 5 - 11 . 
     
     
         13 . The nucleic acid of  claim 22 , wherein the nucleic acid comprises an inducible promoter operably linked to a nucleotide sequence encoding the protein. 
     
     
         14 . An engineered  Lactobacillus  cell comprising the nucleic acid of  claim 12  or  13 . 
     
     
         15 . The engineered  Lactobacillus  cell of  claim 14 , wherein the engineered  Lactobacillus  cell is selected from  L. gasseri  cells and  L. rhamnosus  cells. 
     
     
         16 . A method for producing a protein comprising culturing in cell culture media the engineered  Lactobacillus  cell of  claim 14  or  15  to produce the protein encoded by the nucleic acid. 
     
     
         17 . The method of  claim 16 , wherein the engineered  Lactobacillus  cell is a  Lactobacillus rhamnosus  GG (LGG) cell. 
     
     
         18 . The method of  claim 16  or  17 , wherein the  Lactobacillus  cell produces at least 10 μg of the protein per mL of cell culture media. 
     
     
         19 . The method of any one of  claims 16 - 18  further comprising recovering the protein from the cell culture media. 
     
     
         20 . A method comprising administering to a subject the engineered  Lactobacillus  cell of  claim 14  or  15 . 
     
     
         21 . The method of  claim 20 , wherein the subject has an autoimmune condition. 
     
     
         22 . The method of  claim 21 , wherein the autoimmune condition is inflammatory bowel disease, optionally wherein the inflammatory bowel disease is ulcerative colitis or Crohn's disease. 
     
     
         23 . The method of  claim 20 , wherein the subject has a microbial infection. 
     
     
         24 . A method, comprising:
 (a) computing an amino acid residue position weight matrix (PWM) for a population of signal sequences within a library of bacterial strain-specific sequences; and   (b) generating a consensus signal peptide sequence based on the PWM for the population of signal sequences.   
     
     
         25 . The method of  claim 24  further comprising (c) expressing in cells of the bacterial strain a heterologous protein of interest linked to a signal peptide comprising the consensus signal peptide sequence, optionally wherein the heterologous protein is a therapeutic protein. 
     
     
         26 . A method comprising administering to a subject having an inflammatory bowel disease an engineered  Lactobacillus rhamnosus  cell comprising a nucleic acid encoding an anti-inflammatory cytokine fused to an artificial secretion signal peptide derived from  Lactobacillus rhamnosus.    
     
     
         27 . The method of  claim 26 , wherein the artificial secretion signal peptide comprises an amino acid sequence that has at least 95% identity to an amino acid sequence of SEQ ID NO: 5. 
     
     
         28 . The method of  claim 26 , wherein the artificial secretion signal peptide comprises an amino acid sequence of SEQ ID NO: 5. 
     
     
         29 . The method of any one of  claims 26 - 28 , wherein the anti-inflammatory cytokine is IL10. 
     
     
         30 . The method of any one of  claims 26 - 29 , wherein the nucleic acid comprises an inducible promoter operably linked to a nucleotide sequence encoding the anti-inflammatory cytokine fused to an artificial secretion signal peptide. 
     
     
         31 . The method of any one of  claims 26 - 29 , wherein the amount of anti-inflammatory cytokine produced in the subject by the engineered  Lactobacillus rhamnosus  cell, optionally following a single dose of the engineered  Lactobacillus rhamnosus  cell, is at least 2 times, at least 5 times, or at least 10 times greater than the amount of anti-inflammatory cytokine produced under control conditions, optionally wherein the control conditions include a  Lactobacillus  cell engineered to secrete the anti-inflammatory cytokine. 
     
     
         32 . The method of any one of  claims 26 - 31 , wherein the level of colonic IL10 present in the subject, optionally following a single dose of the engineered  Lactobacillus rhamnosus  cell, is increased by at least 25%, at least 35%, or at least 45%, relative to baseline.