title: Expression and characterization of recombinant human secretory leukocyte protease inhibitor (SLPI) protein from Pichia pastoris.
abstract: The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7kDa peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The post-transformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications.
authors.affiliation: Department of Chemistry, University of the Pacific, Stockton, CA 95211, USA.
authors.forename: Zhiguo
authors.initials: Z
authors.lastname: Li
authors.forename: Allison
authors.initials: A
authors.lastname: Moy
authors.forename: Kirti
authors.initials: K
authors.lastname: Sohal
authors.forename: Carolyn
authors.initials: C
authors.lastname: Dam
authors.forename: Peter
authors.initials: P
authors.lastname: Kuo
authors.forename: James
authors.initials: J
authors.lastname: Whittaker
authors.forename: Mei
authors.initials: M
authors.lastname: Whittaker
authors.forename: Nejat
authors.initials: N
authors.lastname: Düzgünes
authors.forename: Krystyna
authors.initials: K
authors.lastname: Konopka
authors.forename: Andreas H
authors.initials: AH
authors.lastname: Franz
authors.forename: Joan
authors.initials: J
authors.lastname: Lin-Cereghino
authors.forename: Geoff P
authors.initials: GP
authors.lastname: Lin-Cereghino
mesh_terms: D018929:Cell Culture Techniques; D005285:Fermentation; D006031:Glycosylation; D006801:Humans; D010843:Pichia / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D053481:Secretory Leukocyte Peptidase Inhibitor / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection; D014357:Trypsin / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; C506814:SLPI protein, human; D053481:Secretory Leukocyte Peptidase Inhibitor; D014357:Trypsin