Human lysozyme secretion increased by alpha-factor pro-sequence in Pichia pastoris.
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.
Oka
C
C
Chiba Prefectural Industrial Research Institute, Chiba, Japan. coka@iri.pref.chiba.jp
Tanaka
M
M
Muraki
M
M
Harata
K
K
Suzuki
K
K
Jigami
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular / Q000379:methods; D017931:DNA Primers; D006026:Glycoside Hydrolases / Q000235:genetics; D006801:Humans; D000072235:Mating Factor; D008969:Molecular Sequence Data; D009113:Muramidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D010455:Peptides / Q000235:genetics*; D010675:Pheromones / Q000235:genetics; D010843:Pichia; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D011498:Protein Precursors / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D043324:beta-Fructofuranosidase
D017931:DNA Primers; D010455:Peptides; D010675:Pheromones; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000072235:Mating Factor; D006026:Glycoside Hydrolases; D009113:Muramidase; D043324:beta-Fructofuranosidase