title: Expression, purification, and characterization of recombinant human pancreatic duodenal homeobox-1 protein in Pichia pastoris.
abstract: Pancreatic duodenal hemeobox-1 (PDX1) is essential for the development of the embryonic pancreas and plays a key role in pancreatic beta-cell differentiation, maturation, regeneration, and maintenance of normal pancreatic beta-cell insulin-producing function. Purified recombinant PDX1 (rPDX1) may be a useful tool for many research and clinical applications, however, using the Escherichia coli expression system has several drawbacks for producing quality PDX1 protein. To explore the yeast expression system for generating rPDX1 protein, the cDNA coding for the full-length human PDX1 gene was cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rPDX1 was secreted into the culture medium, had a molecular weight by SDS-PAGE of 50kDa, and was glycosylated. The predicted size of the mature unmodified PDX1 polypeptide is 31kDa, suggesting that eukaryotic post-translational modifications are the result of the increased molecular weight. The recombinant protein was purified to greater than 95% purity using a combined ammonium sulfate precipitation with heparin-agarose chromatography. Finally, 120mug of the protein was obtained in high purity from 1L of the culture supernatant. Bioactivity of the rPDX1 was confirmed by the ability to penetrate cell membranes and activation of an insulin-luciferase reporter gene. Our results suggest that the P. pastoris expression system can be used to produce a fully functional human rPDX1 for both research and clinical application.
authors.affiliation: Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
authors.forename: Shi-Wu
authors.initials: SW
authors.lastname: Li
authors.forename: Yuping
authors.initials: Y
authors.lastname: Sun
authors.forename: William
authors.initials: W
authors.lastname: Donelan
authors.forename: Hongfang
authors.initials: H
authors.lastname: Yu
authors.forename: Joanna
authors.initials: J
authors.lastname: Scian
authors.forename: Dongqi
authors.initials: D
authors.lastname: Tang
authors.forename: Li-Jun
authors.initials: LJ
authors.lastname: Yang
mesh_terms: D015153:Blotting, Western; D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006031:Glycosylation; D018398:Homeodomain Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D010843:Pichia / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012394:Rosaniline Dyes; D015534:Trans-Activators / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D018398:Homeodomain Proteins; D011994:Recombinant Proteins; D012394:Rosaniline Dyes; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; C048139:Coomassie blue
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