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Human lysozyme secretion increased by alpha-factor pro-sequence in Pichia pastoris.

C Oka·M Tanaka·M Muraki·K Harata·K Suzuki·Y Jigami
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Abstract

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-alpha Prepro). Transformants harboring a lysozyme gene with MF-alpha 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-alpha Prepro were constructed to discover the function of the pro region. The secretion was dramatically decreased by eliminating the pro region of MF-alpha Prepro. In contrast, MF-alpha 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-alpha Prepro without any spacer sequences was most suitable. The secreted protein by MF-alpha Prepro construct was identical with the authentic human lysozyme, judging from N-terminal amino acid sequencing and molecular mass spectrometric and crystallographic analysis.

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Amino Acid SequenceAnimalsBase SequenceChickensCloning, MolecularDNA PrimersGlycoside HydrolasesHumansMating FactorMolecular Sequence DataMuramidasePeptidesPheromonesPichiaPolymerase Chain ReactionProtein ConformationProtein PrecursorsProtein Sorting SignalsRecombinant ProteinsSaccharomyces cerevisiaeSpectrometry, Mass, Matrix-Assisted Laser Desorption-Ionizationbeta-FructofuranosidaseJournal Article

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C Oka, M Tanaka, M Muraki, K Harata, K Suzuki, Y Jigami. 1999. Human lysozyme secretion increased by alpha-factor pro-sequence in Pichia pastoris.. https://doi.org/10.1271/bbb.63.1977

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