A recombinant rat regenerating protein is mitogenic to pancreatic derived cells.
Pancreatic regenerating protein (reg I) is expressed in acinar cells and is mitogenic to beta- 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-beta-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 beta-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.
Levine
J L
JL
Department of Surgery, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, New York 10461, USA.
Patel
K J
KJ
Zheng
Q h
Qh
Shuldiner
A R
AR
Zenilman
M E
ME
D000595:Amino Acid Sequence / Q000235:genetics; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D051847:Lithostathine; D008934:Mitogens / Q000502:physiology*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins; D010179:Pancreas / Q000166:cytology*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins
D002135:Calcium-Binding Proteins; D051847:Lithostathine; D008934:Mitogens; D009419:Nerve Tissue Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C497616:Reg1a protein, rat