title: Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus.
abstract: BACKGROUND
  Targeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein. These signal sequences usually contain an N-terminal basic amino acid followed by a stretch containing hydrophobic residues, although no consensus signal sequence has been identified. In this study, simple modeling of signal sequences was attempted using Gaussia princeps secretory luciferase (GLuc) in the yeast Kluyveromyces marxianus, which allowed comprehensive recombinant gene construction to substitute synthetic signal sequences.
  
  
  RESULTS
  Mutational analysis of the GLuc signal sequence revealed that the GLuc hydrophobic peptide length was lower limit for effective secretion and that the N-terminal basic residue was indispensable. Deletion of the 16th Glu caused enhanced levels of secreted protein, suggesting that this hydrophilic residue defined the boundary of a hydrophobic peptide stretch. Consequently, we redesigned this domain as a repeat of a single hydrophobic amino acid between the N-terminal Lys and C-terminal Glu. Stretches consisting of Phe, Leu, Ile, or Met were effective for secretion but the number of residues affected secretory activity. A stretch containing sixteen consecutive methionine residues (M16) showed the highest activity; the M16 sequence was therefore utilized for the secretory production of human leukemia inhibitory factor protein in yeast, resulting in enhanced secreted protein yield.
  
  
  CONCLUSIONS
  We present a new concept for the provision of secretory signal sequence ability in the yeast K. marxianus, determined by the number of residues of a single hydrophobic residue located between N-terminal basic and C-terminal acidic amino acid boundaries.
authors.affiliation: Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, 2-16-1 Tokiwadai, Ube, 755-8611, Japan. qqyg6rs9k@arion.ocn.ne.jp.|Present address: Environmental Biofunction Division, National Institute for Agro-Environmental Sciences, 3-1-3 Kan-nondai, Tsukuba, Ibaraki, 305-8604, Japan. qqyg6rs9k@arion.ocn.ne.jp.
authors.forename: Tohru
authors.initials: T
authors.lastname: Yarimizu
authors.affiliation: Innovation Center, Yamaguchi University, 2-16-1 Tokiwadai, Ube, 755-8611, Japan. fukui@yamaguchi-u.ac.jp.
authors.forename: Mikiko
authors.initials: M
authors.lastname: Nakamura
authors.affiliation: Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, 2-16-1 Tokiwadai, Ube, 755-8611, Japan. hoshida@yamaguchi-u.ac.jp.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Hoshida
authors.affiliation: Department of Applied Molecular Bioscience, Yamaguchi University Graduate School of Medicine, 2-16-1 Tokiwadai, Ube, 755-8611, Japan. rinji@yamaguchi-u.ac.jp.
authors.forename: Rinji
authors.initials: R
authors.lastname: Akada
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D060829:Arthropod Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015153:Blotting, Western; D033342:Copepoda / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007716:Kluyveromyces / Q000235:genetics* / Q000378:metabolism; D053605:Leukemia Inhibitory Factor / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid
chemical_list: D000596:Amino Acids; D060829:Arthropod Proteins; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D010455:Peptides; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D008156:Luciferases