Overproduction of human epidermal growth factor/urogastrone in Escherichia coli and demonstration of its full biological activities

Synthesis and secretion of human epidermal growth factor by Escherichia coli.

“…2) was designed and constructed as described (20). The direct-expression vector (pTA1502) and the secretion vector (pTA1522) for the hEGF gene were constructed as shown in Fig.…”

“…Phosphorylated oligodeoxynucleotides (100 pmol) constituting each block were mixed and treated with T4 DNA ligase (800 units) at 14WC overnight to construct block A and block B (Fig. 2) as described (20). The blocks were purified by electrophoresis in 5% polyacrylamide gel, ligated by T4 DNA ligase (500 units), and digested with EcoRI (15 units) 8.0/1 mM EDTA for 10 min at room temperature.…”

“…Cell lysates were prepared by the freeze-thaw method as described (12). The hEGF assay was carried out using human KB cells and 125I-labeled murine EGF (mEGF) as described (20 …”

Production of human epidermal growth factor by an ampicillin resistant recombinant Escherichia coli strain

“…Attempts have been made at the molecular level to optimize EGF expression and product yields (Clements et al, 1991;Engler et al, 1988;Sumi et al, 1985). Cell densities for plasmid-containing Escherichia coli systems expressing foreign proteins are often lower than levels observed with non-plasmid containing E. coli and this has led to studies on optimization of growth of recombinant E. coli (Kracke-Helm et al, 1991;Yee and Blanch, 1992, 1993a, 1993b.…”

Rapid purification and partial characterization of recombinant human epidermal growth factor produced by Escherichia coli

“…Production of medically important human proteins from recombinant microorganisms overcomes supply and extraction problems associated with their production from natural sources. A synthetic hEGF gene has been successfully expressed in yeast and bacterial cells (Smith et al, 1982;Flock er al., 1984;Urdea ec al., 1983;Sumi et al, 1985). We describe here a simple and rapid method for purification of hEGF from a recombinant E. coli fermentation which yields sufficient amounts of this polypeptide for biological and biochemical studies.…”