Modified enterotoxin signal sequences increase secretion level of the recombinant human epidermal growth factor in Escherichia coli.
Amino acid substitutions were made in the heat-labile enterotoxin signal sequence of Escherichia coli by recombinant DNA techniques, and their influence on the secretion of recombinant human epidermal growth factor by E. coli was examined. The heat-labile enterotoxin signal sequence is an amino-terminal extension of the octadecapeptide chain and is comprised of three distinct regions: a positively charged amino-terminal region, a central hydrophobic region, and a carboxyl-terminal region with the cleavage site recognized by the signal peptidase. Some alterations in the signal sequence caused a 1.5-3.5-fold increase in the secretion of recombinant human epidermal growth factor. These were the introduction of: (i) polar and small residues into the carboxyl-terminal region (replacement of Pro-1 Leu-3 with Asn-Ala or Ser-Ala), which may give a favorable structure for the recognition and cleavage by the signal peptidase; and (ii) a polar residue into the central hydrophobic region (replacement of Ile-9 with Ser), which may cause an increase of the affinity to the cytoplasmic membrane. In the latter case, a large amount of the unprocessed "precursor" was accumulated. The combination of these modifications, however, did not work additively. An increase in the amino-terminal positive charge (insertion of Lys) had no effect on secretion. These results prove that the level of protein secretion is greatly dependent on the polarity of the carboxyl-terminal region and the hydrophobicity and/or the amphiphilicity of the central region. Moreover, the overall balance of the physicochemical properties of respective regions is important.
Morioka-Fujimoto
K
K
Biotechnology Research Laboratories, Takeda Chemical Industries Ltd., Osaka, Japan.
Marumoto
R
R
Fukuda
T
T
D000595:Amino Acid Sequence; D001427:Bacterial Toxins / Q000235:genetics*; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism; D004768:Enterotoxins / Q000235:genetics*; D004815:Epidermal Growth Factor / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005822:Genetic Vectors; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; C027538:heat-labile enterotoxin, E coli