High-level production of the mouse epidermal growth factor in a Bacillus brevis expression system.
In order to determine the three-dimensional structure, the folding pathways, and the residues which are critical for biological functions of the mouse epidermal growth factor (mEGF), large amounts of wild-type and site-specific mutants are needed for biological and physiochemical studies. Genes coding for mEGF and its mutants were expressed in a Bacillus brevis system in which the expressed foreign proteins were secreted into the culture medium. However, proteases were also secreted that resulted in partial degradation of the desired foreign proteins. One mutant with much less protease secretion was isolated by applying nitrosoguanidine mutagenesis on a B. brevis strain. A new vector to facilitate DNA mutagenesis and sequencing was also constructed for this system. Methodologies for the transformation of B. brevis and the purification of expressed mEGF were developed. Under the optimized conditions, the production of the mEGF and its mutants was about 50 mg per liter, and yielded up to 10 mg of purified mEGF. Several mEGF mutants have been produced and their receptor binding abilities have been measured.
Wang
B
B
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Yang
X
X
Wu
R
R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001407:Bacillus / Q000235:genetics; D001483:Base Sequence; D010450:Endopeptidases / Q000172:deficiency / Q000235:genetics; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D066246:ErbB Receptors / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D014170:Transformation, Genetic
D000596:Amino Acids; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D010450:Endopeptidases