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Overproduction of human epidermal growth factor/urogastrone in Escherichia coli and demonstration of its full biological activities
Authors:Shin-Ichiro Sumi  Akira Hasegawa  Shintaro Yagi  Ken-ichi Miyoshi  Atsushi Kanezawa  Shizutoshi Nakagawa  Masanori Suzuki
Institution:1. Laboratory of Molecular Biology, The Central Research Laboratories, Wakunaga Pharmaceutical Co. Ltd., Koda, Takada-Gun, Hiroshima 729-64, Japan;2. Laboratory of Pathology, The Central Research Laboratories, Wakunaga Pharmaceutical Co. Ltd., Koda, Takada-Gun, Hiroshima 729-64, Japan
Abstract:A man-made gene coding for 21-Leu] human epidermal growth factor (hEGF)/β-urogastrone was constructed, inserted into a lacZ fusion-gene expression vector, and cloned into Escherichia coli. In the cloned cells the β-galactosidase/hEGF fusion gene was efficiently expressed and the yield of the hybrid protein reached 10% of the total cellular protein. The 21-Leu] hEGF synthesized in the bacterial cells was proved to be as functional as the natural hEGF or urogastrone and mouse EGF by the following criteria: (1) stimulation of cell proliferation, (2) stimulation of thymidine incorporation into cultured cells, (3) competition with mouse EGF for binding sites on the plasma membrane of human KB cells, and (4) stimulation of phosphorylation of a membrane-bound protein of human KB cell, which has an apparent molecular weight of 150 000 to 170 000 and is indistinguishable from the protein phosphorylated in the presence of mouse EGF in the sodium dodecyl sulfate—polyacrylamide electrophoretic gel. The hEGF produced in the bacterial cells also resulted in precocious eyelid-opening by the daily subcutaneous injection into newborn mice and in accelerated incisor eruption in the animals as mouse EGF did, indicating that the hEGF is also fully active in vivo or physiologically.
Keywords:human epidermal growth factor/urogastrone  recombinant DNA  cell proliferation  receptor protein phosphorylation  physiological activities
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