Diphtheria toxin-related alpha-melanocyte-stimulating hormone fusion toxin. Internal in-frame deletion from Thr387 to His485 results in the formation of a highly potent fusion toxin which is resistant to proteolytic degradation.
We have previously reported the genetic construction and properties of a fusion protein which was composed of the enzymatically active and membrane translocation domains of the diphtheria toxin and the receptor-specific ligand alpha-melanocyte-stimulating hormone (alpha-MSH) (Murphy, J.R., Bishai, W., Borowski, M., Miyanohara, A., Boyd, J., and Nagle, S. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 8258-8262). While this fusion toxin was found to be selectively toxic for MSH receptor-bearing cells in vitro, it was subject to profound proteolytic degradation in recombinant Escherichia coli making purification difficult. We now report that the deletion of diphtheria toxin fragment B sequences between Thr387 and His485 results in a protease-resistant form of the fusion toxin, DAB389-alpha-MSH. We show that DAB389-alpha-MSH is expressed in high yield in recombinant Escherichia coli, that it is readily purified from crude bacterial lysates by immunoaffinity and high performance liquid chromatography, and its cytotoxic activity toward both human and murine malignant melanoma cell lines is mediated through the MSH receptor.
Wen
Z L
ZL
Evans Department of Clinical Research, University Hospital, Boston University Medical Center, Massachusetts 02118.
Tao
X
X
Lakkis
F
F
Kiyokawa
T
T
Murphy
J R
JR
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D006224:Cricetinae; D004167:Diphtheria Toxin / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D000882:Haplorhini; D006868:Hydrolysis; D009074:Melanocyte-Stimulating Hormones / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011993:Recombinant Fusion Proteins; D000521:alpha-MSH / Q000235:genetics* / Q000302:isolation & purification / Q000494:pharmacology
D004167:Diphtheria Toxin; D011993:Recombinant Fusion Proteins; C069655:diphtheria toxin-related-alpha-melanocyte-stimulating hormone fusion toxin; D000521:alpha-MSH; D009074:Melanocyte-Stimulating Hormones