title: The effect of α-mating factor secretion signal mutations on recombinant protein expression in Pichia pastoris.
abstract: The methylotrophic yeast, Pichia pastoris, has been genetically engineered to produce many heterologous proteins for industrial and research purposes. In order to secrete proteins for easier purification from the extracellular medium, the coding sequence of recombinant proteins is initially fused to the Saccharomyces cerevisiae α-mating factor secretion signal leader. Extensive site-directed mutagenesis of the prepro-region of the α-mating factor secretion signal sequence was performed in order to determine the effects of various deletions and substitutions on expression. Though some mutations clearly dampened protein expression, deletion of amino acids 57-70, corresponding to the predicted 3rd alpha helix of α-mating factor secretion signal, increased secretion of reporter proteins horseradish peroxidase and lipase at least 50% in small-scale cultures. These findings raise the possibility that the secretory efficiency of the leader can be further enhanced in the future.
authors: Department of Biological Sciences, University of the Pacific Stockton, CA 95211, USA.
authors: Geoff P
authors: GP
authors: Lin-Cereghino
authors: Carolyn M
authors: CM
authors: Stark
authors: Daniel
authors: D
authors: Kim
authors: Jennifer
authors: J
authors: Chang
authors: Nadia
authors: N
authors: Shaheen
authors: Hansel
authors: H
authors: Poerwanto
authors: Kimiko
authors: K
authors: Agari
authors: Pachai
authors: P
authors: Moua
authors: Lauren K
authors: LK
authors: Low
authors: Namphuong
authors: N
authors: Tran
authors: Amy D
authors: AD
authors: Huang
authors: Maria
authors: M
authors: Nattestad
authors: Kristin T
authors: KT
authors: Oshiro
authors: John William
authors: JW
authors: Chang
authors: Archana
authors: A
authors: Chavan
authors: Jerry W
authors: JW
authors: Tsai
authors: Joan
authors: J
authors: Lin-Cereghino
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D017353:Gene Deletion; D015966:Gene Expression Regulation, Fungal; D017930:Genes, Reporter; D006735:Horseradish Peroxidase / Q000235:genetics / Q000378:metabolism; D008049:Lipase / Q000235:genetics / Q000378:metabolism; D000072235:Mating Factor; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids; D060888:Real-Time Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D010455:Peptides; D011994:Recombinant Proteins; D000072235:Mating Factor; D006735:Horseradish Peroxidase; D008049:Lipase