Comparison of His and GST tagged versions of recombinant pancreatitis associated protein 2 in modulation of inflammatory responses.
OBJECTIVE
Pancreatitis associated proteins (PAP) are highly upregulated in acute pancreatitis and other inflammatory states and have been shown to possess immunomodulatory properties. However, continued study of PAP has been hampered by the ability to effectively isolate appropriate amounts of protein from pancreatic juice or efficient generation of recombinant proteins. Here we describe two different methods for generating recombinant PAP2 protein (rPAP2), using either His or GST tagged bacterial methodology with comparison of function.


METHODS
His or GST tagged rPAP2 were generated, cultured with clonal (NR8383) macrophages and compared with respect to inflammatory cytokine expression (IL-1alpha, IL-1beta, IL-6, and TNF-alpha) and bacterial (E. coli) agglutination. Significance was determined by student's t test (P<0.05).


RESULTS
PAP2His treatment induced a 3.6, 2.8, 13.0, 3.5 fold induction of IL-1alpha, IL-1beta, TNF-alpha and IL-6, respectively; similar cytokine expression changes were observed with PAP2GST treatment (3.9, 2.6, 12.2, and 3.0 fold induction of IL-1alpha, IL-1beta, TNF-alpha and IL-6, respectively) (P<0.05). Further, incubation with recombinant PAP2 led to a time dependent increase in bacterial aggregates which was absent in controls.


CONCLUSIONS
These data demonstrate that both methods maintain functional immunomodulatory integrity for PAP2 and provide the ability to generate sufficient quantities to further study structure and function.
Viterbo
Domenico
D
Department of Surgery, SUNY Downstate Medical Center, Brooklyn, NY 11203, USA.
Zenilman
Michael E
ME
Bluth
Martin H
MH
D000951:Antigens, Neoplasm / Q000235:genetics / Q000276:immunology*; D001419:Bacteria / Q000378:metabolism; D014408:Biomarkers, Tumor / Q000235:genetics / Q000276:immunology*; D007249:Inflammation / Q000276:immunology*; D037181:Lectins, C-Type / Q000235:genetics / Q000276:immunology*; D000073718:Pancreatitis-Associated Proteins; D020033:Protein Isoforms / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D037181:Lectins, C-Type; D000073718:Pancreatitis-Associated Proteins; D020033:Protein Isoforms; C000612141:REG3A protein, human; D011993:Recombinant Fusion Proteins
Mol Cell Biol Res Commun. 1999 Sep-Dec;2(3):150-4
10662590
Pancreas. 2009 Jul;38(5):499-502
19342981
J Biol Chem. 1993 Jul 5;268(19):14470-5
8314803
Gastroenterology. 1994 Mar;106(3):728-34
8119544
World J Gastroenterol. 2006 Nov 14;12(42):6806-11
17106929
Infect Immun. 1997 Aug;65(8):3032-6
9234750
Pancreas. 1993 Sep;8(5):597-601
8302796
JOP. 2009 Jan 08;10(1):15-23
19129610
N Engl J Med. 2006 May 18;354(20):2142-50
16707751
J Immunol. 2008 Aug 1;181(3):1959-68
18641333
Protein Expr Purif. 2003 Oct;31(2):197-206
14550637
J Immunol. 2006 Mar 15;176(6):3774-9
16517747
Eur J Biochem. 2000 Mar;267(6):1665-71
10712597
Protein Expr Purif. 2005 May;41(1):98-105
15802226
Pancreas. 2008 May;36(4):402-10
18437087
Protein Eng. 1995 Nov;8(11):1163-9
8819982
J Immunol. 1996 Oct 1;157(7):3139-45
8816425
J Biol Chem. 1991 Dec 25;266(36):24664-9
1722211
Protein Expr Purif. 2009 Nov;68(1):110-20
19464373
J Immunol. 2008 Aug 1;181(3):1948-58
18641332
Protein Expr Purif. 2006 Jul;48(1):151-9
16504538
Gut. 2007 Aug;56(8):1091-7
17409121
Eur J Biochem. 1995 Jun 1;230(2):503-10
7607222
Ann Surg. 2000 Nov;232(5):646-52
11066135
J Neurosci. 2006 Jul 12;26(28):7460-7
16837594
J Clin Invest. 1992 Dec;90(6):2284-91
1469087
BMC Surg. 2005 Oct 20;5:22
16242024
Scand J Gastroenterol. 2004 Sep;39(9):870-81
15513386