Synthetic signal sequences that enable efficient secretory protein production in the yeast Kluyveromyces marxianus.

Tohru Yarimizu,Rinji Akada,Mikiko Nakamura,Hisashi Hoshida

doi:10.1186/s12934-015-0203-y

Tohru Yarimizu, Rinji Akada + Show 2 more

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https://doi.org/10.1186/s12934-015-0203-y

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Abstract

BackgroundTargeting 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.ResultsMutational 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0203-y) contains supplementary material, which is available to authorized users.

Highlights

Targeting of cellular proteins to the extracellular environment is directed by a secretory signal sequence located at the N-terminus of a secretory protein
The results indicated that the number of amino acids adequate for efficient secretion could be defined when a single hydrophobic amino acid repeat was utilized instead
The N-terminal 17 amino acid sequence was indicated as the signal sequence of Gaussia princeps secretory luciferase (GLuc) (New England Bio Labs, Inc.)

Summary

Introduction

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, no consensus signal sequence has been identified. The signal sequence for a secretory protein is the first designated peptide sequence that exhibits similarity with the common amino-acid domain located at the N-terminus of all secretory proteins [1,2,3]. The signal peptide usually consists of an N-terminal basic residue and a subsequent stretch of amino acids containing a hydrophobic core, known to be recognized by the signal recognition particle in both prokaryotes and eukaryotes.

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