Construction of a bifunctional Escherichia coli heat-stable enterotoxin (STb)-alkaline phosphatase fusion protein.
A fusion between the genes encoding the Escherichia coli STb heat-stable enterotoxin (estB) and alkaline phosphatase (phoA) was constructed, and the expressed protein product was characterized. The STb-alkaline phosphatase protein (STb-PhoA) had an apparent molecular mass of 50,000 daltons and was detected with both monoclonal anti-alkaline phosphatase and polyclonal anti-STb antibodies. Expression of the gene fusion resulted in high-level production of alkaline phosphatase activity, indicating that STb-PhoA was processed and exported into the periplasm of the E. coli host strain. Amino acid sequence analysis of the hybrid protein yielded the sequence Ser-Thr-Gln-Ser-Asn-Lys-Lys, indicating that STb-PhoA was processed during export in a fashion identical to that of native STb (Y. M. Kupersztoch, K. Tachias, C. R. Moomaw, L. A. Dreyfus, R. G. Urban, C. Slaughter, and S. Whipp, J. Bacteriol. 172: 2427-2432, 1990). STb-PhoA was purified from an expressed bacterial lysate by preparative isoelectric focusing. In a rat ligated intestinal loop model, purified STb-PhoA induced highly significant (P less than 0.002) fluid secretion. In addition, the specific activity of STb-PhoA was nearly identical to that of purified STb. Thus, the STb-PhoA hybrid protein represents a readily obtainable source of biologically active (STb) enterotoxin that may prove useful in studies to determine the mode of toxin action.
Urban
R G
RG
Department of Microbiology, University of Texas Medical Branch, Galveston 77550-2782.
Dreyfus
L A
LA
Whipp
S C
SC
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics*; D001435:Bacteriophages / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D029968:Escherichia coli Proteins; D007413:Intestinal Mucosa / Q000378:metabolism; D008969:Molecular Sequence Data; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics
D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; C034151:heat stable toxin (E coli); D000469:Alkaline Phosphatase