title: Isolation of Pichia pastoris PIR genes and their utilization for cell surface display and recombinant protein secretion.
abstract: Proteins with internal repeats are highly conserved among budding yeasts. In this study, the isolation of two proteins with internal repeats (PIR) genes, i.e. PpPIR1 and PpPIR2, from the methylotrophic yeast Pichia pastoris has been reported. The PIR1 and PIR2 genes' open reading frames were found to contain 1068 and 972 bases, respectively. The sequence homology search showed a homologous conserved repeat of PIR yeast block (SQIGDGQIQATT) in both proteins. The PIR yeast block was present eight times in the PpPir1p and four times in the PpPir2p proteins. Both proteins showed conserved glutamine (Q) and aspartic acid (D) in the repeated sequences, indicating a possible alkali-sensitive β1,3-glucan ester linkage. The fusion constructs of PpPir1-2p and enhanced green fluorescent protein (EGFP) were developed for yeast cell surface display. The immunofluorescence assay showed uniform localization of EGFP protein on the P. pastoris cell surface in all fusion constructs. Furthermore, new vectors were developed for recombinant protein secretion in P. pastoris, utilizing the pre-pro signal of PpPir1p protein. Efficient processing of the signal sequence was observed from EGFP and human α1-antitrypsin (AAT) fusion constructs and recombinant protein secretion was obtained in the culture supernatant.
authors.affiliation: Biological Process Development Facility, Department of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, NE 68588, USA.
authors.forename: Yogender Pal
authors.initials: YP
authors.lastname: Khasa
authors.forename: Sara
authors.initials: S
authors.lastname: Conrad
authors.forename: Mustafa
authors.initials: M
authors.lastname: Sengul
authors.forename: Sarah
authors.initials: S
authors.lastname: Plautz
authors.forename: Michael M
authors.initials: MM
authors.lastname: Meagher
authors.forename: Mehmet
authors.initials: M
authors.lastname: Inan
mesh_terms: D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D005455:Fluorescent Antibody Technique; D005470:Fluorometry; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D017930:Genes, Reporter; D005827:Genetics, Microbial / Q000379:methods*; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D017422:Sequence Analysis, DNA
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D006023:Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins