Structure-function studies of murine epidermal growth factor: expression and site-directed mutagenesis of epidermal growth factor gene.
Wild-type murine epidermal growth factor (mEGF) and mutants with Leu47 replaced by serine and valine, respectively, have been produced by recombinant DNA methodology. A synthetic gene for mEGF was fused to the coding sequence for the signal peptide of the outer membrane protein A (ompA) of Escherichia coli in the secretion vector pIN-III-ompA3, and the recombinant plasmid was used to transform E. coli. Upon induction of gene expression, mEGF and the mutants was expressed and secreted into the periplasmic space. Purification of the wild-type Leu47-mEGF and the mutants was carried out by reversed-phase and anion-exchange high-performance liquid chromatography (HPLC). Amino acid analysis and Western blot analysis further confirmed the identities of the proteins. Specific activities for wild-type and mutant proteins were measured in both mEGF receptor binding and autophosphorylation assays. The recombinant mEGF has specific activities identical with that of mEGF purified from mouse submaxillary glands, while both mutants have reduced specific activities in both bioassays. The data demonstrate the importance of the highly conserved Leu47 residue in mEGF for full biological activity.
Ray
P
P
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, New York 14853.
Moy
F J
FJ
Montelione
G T
GT
Liu
J F
JF
Narang
S A
SA
Scheraga
H A
HA
Wu
R
R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D015153:Blotting, Western; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004815:Epidermal Growth Factor / Q000235:genetics* / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D007930:Leucine; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine; D013329:Structure-Activity Relationship; D014633:Valine
D000596:Amino Acids; D011994:Recombinant Proteins; D012694:Serine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D004262:DNA Restriction Enzymes; D007930:Leucine; D014633:Valine