Expression and characterization of recombinant human secretory leukocyte protease inhibitor (SLPI) protein from Pichia pastoris.
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.
Li
Zhiguo
Z
Department of Chemistry, University of the Pacific, Stockton, CA 95211, USA.
Moy
Allison
A
Sohal
Kirti
K
Dam
Carolyn
C
Kuo
Peter
P
Whittaker
James
J
Whittaker
Mei
M
Düzgünes
Nejat
N
Konopka
Krystyna
K
Franz
Andreas H
AH
Lin-Cereghino
Joan
J
Lin-Cereghino
Geoff P
GP
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
D011994:Recombinant Proteins; C506814:SLPI protein, human; D053481:Secretory Leukocyte Peptidase Inhibitor; D014357:Trypsin