A recombinant rat regenerating protein is mitogenic to pancreatic derived cells

Joshua L. Levine, Ketul J. Patel, Qing hu Zheng, Alan R. Shuldiner, Michael E. Zenilman

Research output: Contribution to journalArticle

Abstract

Pancreatic regenerating protein (reg I) is expressed in acinar cells and is mitogenic to β- and ductal cells. Isolation of large amounts of endogenous reg I for in vivo and in vitro experiments has been difficult. The aim of this study was to develop a recombinant protein and determine its bioactivity on rat pancreatic derived cells. cDNA of the rat reg I coding region was created with unique BamHI flanking sequences using reverse transcriptase PCR. The coding region was then cloned into a bacterial expression vector in which expression is controlled by a T7 promoter. After transformation into the Escherichia coli strain B21(DE3) and induction by isopropyl-β-D-thiogalactopyranoside, a fusion protein of 24 kDa in size, named reg-PET, was noted in the bacterial lysate. This protein contained a polyhistidine and S-peptide sequence to facilitate isolation and identification, respectively. This protein was purified using affinity chromatography, and identity was confirmed with gel electrophoresis and Western analysis. The reg-PET protein was mitogenic to both ARIP and RIN cells, rat pancreatic ductal and β-cell lines, respectively. Antibodies raised to the protein reacted against rat reg I in pancreas. The purified recombinant reg I fusion protein, like endogenous reg I, is mitogenic to pancreatic derived cells. It is more potent than reg I isolated from pancreatic tissue. This protein can be isolated rapidly, easily, and with a high amount of purity. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)60-65
Number of pages6
JournalJournal of Surgical Research
Volume89
Issue number1
DOIs
StatePublished - Mar 2000

ASJC Scopus subject areas

  • Surgery

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    Levine, J. L., Patel, K. J., Zheng, Q. H., Shuldiner, A. R., & Zenilman, M. E. (2000). A recombinant rat regenerating protein is mitogenic to pancreatic derived cells. Journal of Surgical Research, 89(1), 60-65. https://doi.org/10.1006/jsre.1999.5800