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 Escherich...

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Bibliographic Details
Published in:Biochemistry (Easton) 1988-09, Vol.27 (19), p.7289-7295
Main Authors: Ray, Prabir, Moy, Franklin J, Montelione, Gaetano T, Liu, Jin Fu, Narang, Saran A, Scheraga, Harold A, Wu, Ray
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Amino Acids - analysis
Analytical, structural and metabolic biochemistry
Animals
Base Sequence
Binding, Competitive
Biological and medical sciences
Blotting, Western
Cell Line, Transformed
Cell Membrane - metabolism
Cloning, Molecular
DNA Restriction Enzymes
Epidermal Growth Factor - genetics
Epidermal Growth Factor - metabolism
ErbB Receptors - metabolism
Escherichia coli - genetics
Fundamental and applied biological sciences. Psychology
Gene Expression Regulation
Leucine
Mice
Molecular Sequence Data
Mutation
Protein hormones. Growth factors. Cytokines
Proteins
Recombinant Proteins - metabolism
Serine
Structure-Activity Relationship
Valine
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Summary: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.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi00419a017