Human Lysozyme Secretion Increased by Alpha-factor Pro-sequence in Pichia pastoris

• Published in: Bioscience, biotechnology, and biochemistry Vol. 63; no. 11; pp. 1977 - 1983
• Main Authors: OKA, Chitoshi, TANAKA, Masao, MURAKI, Michiro, HARATA, Kazuaki, SUZUKI, Katsunori, JIGAMI, Yoshifumi
• Format: Journal Article
• Language: English
• Published: Tokyo Japan Society for Bioscience, Biotechnology, and Agrochemistry 01.11.1999; Japan Society for Bioscience Biotechnology and Agrochemistry; Oxford University Press
• Subjects: alpha factor; Amino Acid Sequence; Animals; Base Sequence; beta-Fructofuranosidase; Biological and medical sciences; Biotechnology; Chickens; Cloning, Molecular - methods; DNA Primers; Fundamental and applied biological sciences. Psychology; Genetic engineering; Genetic technics; Glycoside Hydrolases - genetics; human lysozyme; Humans; Mating Factor; Methods. Procedures. Technologies; Modification of gene expression level; Molecular Sequence Data; Muramidase - biosynthesis; Muramidase - chemistry; Muramidase - genetics; Peptides - genetics; Pheromones - genetics; Pichia; Pichia pastoris; Polymerase Chain Reaction; Protein Conformation; Protein Precursors - genetics; Protein Sorting Signals - genetics; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Saccharomyces; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; secretion; signal peptides; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Human; Leader peptide; Yeast; Leader sequence; Enzyme; Secretion; Gene expression; Fungi; Glycosidases; Hydrolases; Ascomycetes; Recombinant protein; O-Glycosidases; Hybrid gene; Lysozyme; Thallophyta
• ISSN: 0916-8451; 1347-6947
• DOI: 10.1271/bbb.63.1977

To get high level secretion of human lysozyme in Pichia pastoris, the following three signal sequences and one prepro sequence were evaluated: chicken lysozyme signal peptide, leucine-rich artificial signal peptide, Saccharomyces invertase signal peptide, and Saccharomyces prepro sequence of alpha factor (MF-α Prepro). Transformants harboring a lysozyme gene with MF-α Prepro secreted 20-fold more lysozyme than those harboring the lysozyme gene with any one of the other three signal sequences. Three mutant leader sequences derived from MF-α Prepro were constructed to discover the function of the pro region. The secretion was dramatically decreased by eliminating the pro region of MF-α Prepro. In contrast, MF-α Prepro with the EAEAEA sequence directed the secretion of an equivalent level of lysozyme having the extra amino acids (EAEAEA) in its N-terminus. For the effective secretion of native human lysozyme, MF-α Prepro without any spacer sequences was most suitable. The secreted protein by MF-α Prepro construct was identical with the authentic human lysozyme, judging from N-terminal amino acid sequencing and molecular mass spectrometric and crystallographic analysis.