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 prom oter, the signal sequence of Pseudomonas shutzeri amylase, and the syn thesized EGF gene into the broad-host-range vector pKT230. E. coli JM1 09 carrying pKSEGP2 secreted EGF into the periplasm and the culture me dium under the control of the tac promoter. Pseudomonas aeruginosa PAO 1161 carrying pKSEGF2 and Pseudomonas putida AC10 carrying pKSEGF2 sec reted EGF into the culture medium constitutively. Four hydrogen-utiliz ing bacteria, Pseudomonas pseudoflava, Alcaligenes eutrophus, Alcalige nes paradoxus, and Paracoccus denitrificans, were transconjugated with pKSEGF2 from eight hydrogen-utilizing bacteria tested. In these trans conjugated hydrogen-utilizing bacteria, P. pseudoflava carrying pKSEGF 2 grew autotrophically and secreted EGF, confirmed by Western blot (im munoblot) analysis, into the culture medium constitutively.