TY - JOUR
T1 - A recombinant rat regenerating protein is mitogenic to pancreatic derived cells
AU - Levine, Joshua L.
AU - Patel, Ketul J.
AU - Zheng, Qing hu
AU - Shuldiner, Alan R.
AU - Zenilman, Michael E.
N1 - Funding Information:
This study was supported by NIH RO1 DK 54511-01 and Core Grant P60 DK20541. The DNA Sequencing Facility at Albert Einstein College of Medicine is supported by NCI Cancer Research Center Core Support Grant CA13330.
PY - 2000/3
Y1 - 2000/3
N2 - 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.
AB - 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.
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U2 - 10.1006/jsre.1999.5800
DO - 10.1006/jsre.1999.5800
M3 - Article
C2 - 10720454
AN - SCOPUS:0034049604
SN - 0022-4804
VL - 89
SP - 60
EP - 65
JO - Journal of Surgical Research
JF - Journal of Surgical Research
IS - 1
ER -