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

概要

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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 (ME-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.
社団法人日本農芸化学会の論文
1999-11-23