Signal peptide optimization tool for the secretion of recombinant protein from Saccharomyces cerevisiae.
The secretion efficiency of foreign proteins in recombinant microbes is strongly dependent on the combination of the signal peptides (SPs) used and the target proteins; therefore, identifying the optimal SP sequence for each target protein is a crucial step in maximizing the efficiency of protein secretion in both prokaryotes and eukaryotes. In this study, we developed a novel method, named the SP optimization tool (SPOT), for the generation and rapid screening of a library of SP-target gene fusion constructs to identify the optimal SP for maximizing target protein secretion. In contrast to libraries generated in previous studies, SPOT fusion constructs are generated without adding the intervening sequences associated with restriction enzyme digestion sites. Therefore, no extra amino acids are inserted at the N-terminus of the target protein that might affect its function or conformational stability. As a model system, β-galactosidase (LacA) from Aspergillus oryzae was used as a target protein for secretion from Saccharomyces cerevisiae. In total, 60 SPs were selected from S. cerevisiae secretory proteins and utilized to generate the SP library. While many of the SP-LacA fusions were not secreted, several of the SPs, AGA2, CRH1, PLB1, and MF(alpha)1, were found to enhance LacA secretion compared to the WT sequence. Our results indicate that SPOT is a valuable method for optimizing the bioproduction of any target protein, and could be adapted to many host strains.
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Akihiro
A
Mori
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Shoichi
S
Hara
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Tomohiro
T
Sugahara
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Takaaki
T
Kojima
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Yugo
Y
Iwasaki
Biomolecular Engineering Laboratory, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.
Yasuaki
Y
Kawarasaki
Bioproduction Research Institute (BPRI), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba 305-8566, Japan.
Takehiko
T
Sahara
Bioproduction Research Institute (BPRI), National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-higashi, Toyohira-ku, Sapporo 062-8517, Japan.
Satoru
S
Ohgiya
Laboratory of Molecular Biotechnology, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Electronic address: hnakano@agr.nagoya-u.ac.jp.
Hideo
H
Nakano
D000595:Amino Acid Sequence; D004262:DNA Restriction Enzymes / Q000378:metabolism; D008969:Molecular Sequence Data; D019151:Peptide Library; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology*; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D001616:beta-Galactosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D019151:Peptide Library; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D004262:DNA Restriction Enzymes; D001616:beta-Galactosidase
Saccharomyces cerevisiae; Secretion; Signal peptide library; SignalP; β-Galactosidase