Expression, purification, and characterization of recombinant human pancreatic duodenal homeobox-1 protein in Pichia pastoris.
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.
Li
Shi-Wu
SW
Department of Pathology, Immunology, and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA.
Sun
Yuping
Y
Donelan
William
W
Yu
Hongfang
H
Scian
Joanna
J
Tang
Dongqi
D
Yang
Li-Jun
LJ
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
D018398:Homeodomain Proteins; D011994:Recombinant Proteins; D012394:Rosaniline Dyes; D015534:Trans-Activators; C084010:pancreatic and duodenal homeobox 1 protein; C048139:Coomassie blue
Biotechnol Bioeng. 2002 Sep 30;79(7):713-23
12209794
Nature. 1994 Oct 13;371(6498):606-9
7935793
Curr Biol. 2008 Jun 24;18(12):890-4
18501604
Biochim Biophys Acta. 2005 Aug 31;1752(1):18-25
16112628
Diabetologia. 2002 Mar;45(3):309-26
11914736
J Clin Invest. 2002 May;109(9):1193-201
11994408
Proc Natl Acad Sci U S A. 2009 Sep 15;106(37):15768-73
19720998
Diabetes. 2005 Sep;54(9):2586-95
16123346
Nat Genet. 1997 Jan;15(1):106-10
8988180
Front Biosci. 2008 May 01;13:6170-82
18508651
Protein Expr Purif. 2009 Oct;67(2):175-81
19505578
Proc Natl Acad Sci U S A. 2002 Sep 17;99(19):12236-41
12221286
Biotechnol Bioeng. 2004 Feb 5;85(3):340-50
14748090
Annu Rev Biochem. 1996;65:271-303
8811181
Biochem Biophys Res Commun. 2005 Jun 24;332(1):68-74
15896300
J Biol Chem. 1999 Jan 8;274(2):1011-6
9873045
Diabetes. 2003 Jul;52(7):1732-7
12829640
Acta Biochim Biophys Sin (Shanghai). 2009 Nov;41(11):900-9
19902124
Biotechnol Prog. 2006 Jul-Aug;22(4):1090-5
16889384
Biochem Biophys Res Commun. 2008 May 30;370(2):225-9
18361919
Diabetes. 1999 Mar;48(3):507-13
10078550
Differentiation. 2001 Oct;68(4-5):205-19
11776473
Arch Biochem Biophys. 2003 Jul 15;415(2):155-63
12831837
Biotechnol Prog. 2007 Mar-Apr;23(2):364-9
17315887
J Biol Chem. 1996 Nov 29;271(48):30299-302
8939984
Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15057-62
8986763
Diabetes. 2005 Oct;54(10):2859-66
16186386
Appl Microbiol Biotechnol. 2010 Mar;86(1):243-53
19826807
Biochem Soc Trans. 2007 Aug;35(Pt 4):811-5
17635154
Biotechnol Adv. 2009 May-Jun;27(3):297-306
19500547
Eur J Endocrinol. 2002 Feb;146(2):129-41
11834421
Stem Cells. 2003;21(1):111-7
12529558
BMC Res Notes. 2009 Jan 09;2:3
19134185
Mol Cell Biol. 2002 Jan;22(2):412-20
11756538
Mol Ther. 2003 Feb;7(2):281-7
12597917
J Periodontol. 2006 Aug;77(8):1289-303
16881798
J Mol Recognit. 2005 Mar-Apr;18(2):119-38
15565717
Diabetes. 2008 Mar;57(3):757-69
18086901