Secretion of human epidermal growth factor (EGF) in autotrophic culture by a recombinant hydrogen-utilizing bacterium, Pseudomonas pseudoflava, carrying broad-host-range EGF secretion vector pKSEGF2.
We constructed the broad-host-range human epidermal growth factor (EGF) secretion plasmid pKSEGF2 by inserting the Escherichia coli tac promoter, the signal sequence of Pseudomonas stutzeri amylase, and the synthesized EGF gene into the broad-host-range vector pKT230. E. coli JM109 carrying pKSEGF2 secreted EGF into the periplasm and the culture medium under the control of the tac promoter. Pseudomonas aeruginosa PAO1161 carrying pKSEGF2 and Pseudomonas putida AC10 carrying pKSEGF2 secreted EGF into the culture medium constitutively. Four hydrogen-utilizing bacteria, Pseudomonas pseudoflava, Alcaligenes eutrophus, Alcaligenes paradoxus, and Paracoccus denitrificans, were transconjugated with pKSEGF2 from eight hydrogen-utilizing bacteria tested. In these transconjugated hydrogen-utilizing bacteria, P. pseudoflava carrying pKSEGF2 grew autotrophically and secreted EGF, confirmed by Western blot (immunoblot) analysis, into the culture medium constitutively.
Hayase
N
N
Biotechnology Research Laboratory, Kobe Steel Ltd., Hyogo, Japan.
Ishiyama
A
A
Niwano
M
M
D000421:Alcaligenes / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002245:Carbon Dioxide / Q000378:metabolism; D003227:Conjugation, Genetic; D004815:Epidermal Growth Factor / Q000096:biosynthesis* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005632:Fructose / Q000378:metabolism; D005822:Genetic Vectors; D005947:Glucose / Q000378:metabolism; D006801:Humans; D006859:Hydrogen / Q000378:metabolism; D008969:Molecular Sequence Data; D010230:Paracoccus / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D011549:Pseudomonas / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*
D021382:Protein Sorting Signals; D002245:Carbon Dioxide; D005632:Fructose; D004815:Epidermal Growth Factor; D006859:Hydrogen; D005947:Glucose