Secreted expression of human lysozyme in the yeast Pichia pastoris under the direction of the signal peptide from human serum albumin.
hLM (human lysozyme) has important potential application as a future safely administered human drug and food additive. To produce secreted rhLM (recombinant hLM) from the yeast Pichia pastoris, the signal peptide from HSA (human serum albumin) was employed to direct secreted expression. On the basis of the vector pPIC3.5k, an overexpression vector, pPIC3.5k-hLM, carrying the strong promoter AOX1 (aldehyde oxidase 1), the HSA signal peptide, the enterokinase recognition motif, the lysozyme gene and other necessary genetic segments was constructed and this was followed by a series of genetic manipulations. A positive colony was picked off to test its expression pattern. The target protein, rhLM, was obtained from the supernatant and showed a gradual enrichment with the induction time course, reaching its highest level at 72 h. This pattern was identical with that shown by the secreted expression of a heterologous protein directed by Saccharomcyes cerevisiae a-mating factor prepro-signal peptide in P. pastoris. After a series of purification processes, including ultrafiltration with a hollow-fibre membrane module, DEAE-Sepharose, Sephadex G50 chromatography and enterokinase digestion, the mature protein was characterized by MALDI-TOF-MS/MS (matrix-assisted laser-desorption ionization-time-of-flight tandem MS), N-terminal amino acid sequencing, and K(m) and K(cat) determination. The results confirmed that the rhLM was identical with native hLM. Moreover, the mature protein exhibited in vitro bacteriolytic activity against the Gram-positive bacterium Micrococcus lysodeikticus and the Gram-negative bacterium Escherichia coli. Taken together, it appeared that the HSA signal peptide was able direct secretive expression of a heterologous protein in P. pastoris.
Xiong
Runsong
R
College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, People's Republic of China.
Chen
Jingjing
J
Chen
Jinchun
J
D000595:Amino Acid Sequence; D016384:Consensus Sequence; D015870:Gene Expression; D005818:Genetic Engineering; D006801:Humans; D000072235:Mating Factor; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D021382:Protein Sorting Signals; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D017422:Sequence Analysis, DNA; D012709:Serum Albumin / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D010455:Peptides; D021382:Protein Sorting Signals; D012709:Serum Albumin; D000072235:Mating Factor; D009113:Muramidase