OBJECTIVE AND DESIGN: 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.
OBJECTIVE AND DESIGN: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.
Authors: Domenico Viterbo; Gordon E Callender; Theresa DiMaio; Cathy M Mueller; Tamar Smith-Norowitz; Michael E Zenilman; Martin H Bluth Journal: JOP Date: 2009-01-08