title: Structural characterization of the α-mating factor prepro-peptide for secretion of recombinant proteins in Pichia pastoris.
abstract: The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion.
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Sabreen
authors.initials: S
authors.lastname: Chahal
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Peter
authors.initials: P
authors.lastname: Wei
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Pachai
authors.initials: P
authors.lastname: Moua
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Sung Pil James
authors.initials: SP
authors.lastname: Park
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Janet
authors.initials: J
authors.lastname: Kwon
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Arth
authors.initials: A
authors.lastname: Patel
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Anthony T
authors.initials: AT
authors.lastname: Vu
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Catolico
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Yu Fang Tina
authors.initials: YF
authors.lastname: Tsai
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Nadia
authors.initials: N
authors.lastname: Shaheen
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Tiffany T
authors.initials: TT
authors.lastname: Chu
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Vivian
authors.initials: V
authors.lastname: Tam
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Zill-E-Huma
authors.initials: ZE
authors.lastname: Khan
authors.affiliation: Department of Chemistry, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Hyun Henry
authors.initials: HH
authors.lastname: Joo
authors.affiliation: Department of Chemistry, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Xue
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Joan
authors.initials: J
authors.lastname: Lin-Cereghino
authors.affiliation: Department of Chemistry, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Jerry W
authors.initials: JW
authors.lastname: Tsai
authors.affiliation: Department of Biological Sciences, University of the Pacific, Stockton, CA 95211, USA. Electronic address: glincere@pacific.edu.
authors.forename: Geoff P
authors.initials: GP
authors.lastname: Lin-Cereghino
mesh_terms: D000595:Amino Acid Sequence; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006735:Horseradish Peroxidase / Q000235:genetics / Q000378:metabolism; D008049:Lipase / Q000235:genetics / Q000378:metabolism; D000072235:Mating Factor / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion
chemical_list: D005656:Fungal Proteins; D010455:Peptides; D011498:Protein Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D000072235:Mating Factor; D006735:Horseradish Peroxidase; D008049:Lipase; C431349:lipase B, Candida antarctica
keywords: Alpha mating factor; Circular dichroism; Knob socket; Pichia pastoris; Protein modeling; Recombinant expression; Secretion leader