Comparison of His and GST tagged versions of recombinant pancreatitis associated protein 2 in modulation of inflammatory responses

Viterbo, Domenico; Zenilman, Michael E.; Bluth, Martin H.

doi:10.1007/s00011-010-0194-4

Cited by 2 publications

(3 citation statements)

References 29 publications

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“…The generation of wild-type and modified Reg proteins in a variety of hosts including E. coli (Lee et al, 2003; Cash et al, 2006), yeast (Li et al, 2003), mammalian (HEK293 or CHO) cells (Namikawa et al, 2006) and even transgenic mice (Christa et al, 2000) has aided significantly the research on various aspects of Reg protein biology. For instance, recombinant rat PAP2 protein produced in E. coli with either a GST or 6xHis tag (Viterbo et al, 2010) is detected by western blotting at the expected size, mediates bacterial aggregation and causes the upregulation of interleukins (IL-1α, -1β and -6) and TNF-α genes in rat NR8383 macrophages. It should be noted that various Reg members (e.g., lithostathine) feature glycosylation moieties, which may contribute to the protein function and therefore bacterial (lacking glycosylation) and yeast (typically mannose-rich glycosylation) systems may not be ideal for the production of recombinant Reg products with physiological activity.…”

Section: Challengesmentioning

confidence: 99%

Four Decades After the Discovery of Regenerating Islet-Derived (Reg) Proteins: Current Understanding and Challenges

Chen

Downing

Tzanakakis

2019

Front. Cell Dev. Biol.

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Regenerating islet-derived (Reg) proteins have emerged as multifunctional agents with pro-proliferative, anti-apoptotic, differentiation-inducing and bactericidal properties. Over the last 40 years since first discovered, Reg proteins have been implicated in a gamut of maladies including diabetes, various types of cancer of the digestive tract, and Alzheimer disease. Surprisingly though, a consensus is still absent on the regulation of their expression, and molecular underpinning of their function. Here, we provide a critical appraisal of recent findings in the field of Reg protein biology. Specifically, the structural characteristics are reviewed particularly in connection with established or purported functions of different members of the Reg family. Moreover, Reg expression patterns in different tissues both under normal and pathophysiological conditions are summarized. Putative receptors and cascades reported to relay Reg signaling inciting cellular responses are presented aiming at a better appreciation of the biological activities of the distinct Reg moieties. Challenges are also discussed that have hampered thus far the rapid progress in this field such as the use of non-standard nomenclature for Reg molecules among various research groups, the existence of multiple Reg members with significant degree of homology and possibly compensatory modes of action, and the need for common assays with robust readouts of Reg activity. Coordinated research is warranted going forward, given that several research groups have independently linked Reg proteins to diseased states and raised the possibility that these biomolecules can serve as therapeutic targets and biomarkers.

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Section: Challengesmentioning

confidence: 99%

Four Decades After the Discovery of Regenerating Islet-Derived (Reg) Proteins: Current Understanding and Challenges

Chen

Downing

Tzanakakis

2019

Front. Cell Dev. Biol.

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“…Studies to clarify the function of PAP have been limited due to the problems in isolation and production of PAP isoforms. This is largely due to the low solubility of PAP isoforms in the liquid phase (Viterbo et al, 2010). With this study, the production of recombinant PAP protein, which has many uses, has been described using the K. phaffii X33 yeast strain.…”

Section: Introductionmentioning

confidence: 99%

“…Biophysically, the Fc region folds independently and can improve the solubility and stability of the co-molecule both in vitro and in vivo (Carter, 2011). Previous studies show that the solubility of PAP is limited (Viterbo et al, 2010). Therefore, the recombinant PAP protein was produced as fused to the Fc-tag both to facilitate the purification of the PAP protein and to increase its solubility.…”

Section: Introductionmentioning

confidence: 99%

Production of Human Pancreatitis-Associated Protein (hPAP) in Komagataella phaffii (Pichia pastoris)

Pazarli

Yücel

Akçael

et al. 2021

GenApp

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Pancreatitis-associated protein (PAP) is a pancreatic stress protein that is not produced in a healthy pancreas but is highly synthesized in pancreatic acinar cells in response to acute and chronic pancreatitis, hypoxia, toxins, diabetes, lipopolysaccharides hypotransferrinemia and organ transplantation. Changes in the PAP levels in serum are an important biological marker in the early stage of pancreatic diseases. In this study, the recombinant human PAP protein, which has the potential to be used as a diagnostic marker and as research material in proliferation, apoptosis, cell migration, cell invasion, and immunoassay studies, was expressed efficiently under the control of the AOX1 gene promoter in the Komagataella phaffii (Pichia pastoris) (K. phaffii) X33 strain. We describe the conditions required for the efficient production of PAP protein by methanol induction and its use without purification. The produced unpurified protein was tested in sandwich ELISA and showed consistent results with the commercial product. These results are encouraging that the protein produced can be used as a biomarker standard in ELISA tests without the cost and labor of purification.

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Comparison of His and GST tagged versions of recombinant pancreatitis associated protein 2 in modulation of inflammatory responses

Abstract: 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.

Cited by 2 publications

References 29 publications

Four Decades After the Discovery of Regenerating Islet-Derived (Reg) Proteins: Current Understanding and Challenges

Four Decades After the Discovery of Regenerating Islet-Derived (Reg) Proteins: Current Understanding and Challenges

Production of Human Pancreatitis-Associated Protein (hPAP) in Komagataella phaffii (Pichia pastoris)

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