Overproduction of soluble, extracellular cytotoxin α-sarcin inEscherichia coli
- 58 Downloads
The goal of the present study was to establish the condition to obtain preparative amounts of the recombinant cytotoxin α-sarcin to be used for immunoconjugate production.
α-Sarcin cDNA was isolated fromAspergillus giganteus strain MDH 18894 and its expression inEscherichia coli was attempted by the use of both two-cistron and fusion protein-expression systems. Whereas the former resulted in low intracellular expression level of recombinant α-sarcin (r-Sar), the latter allowed high-level expression of the fusion protein in the culture supernant.
A variant form of α-sarcin with an additional threonine residue in position 1 (Thr-Sar) was obtained by proteolytic processing of the fusion protein with a final yield after purification of 40 mg/L of culture. Both recombinant proteins r-Sar and Thr-Sar were identical to native a-sarcin with respect to the biochemical properties and to the in vitro biological activity.
Index EntriesRibosome-inactivating protein Aspergillus giganteus toxin E. coli expression recombinant protein secretion
Unable to display preview. Download preview PDF.
- 5.Parente, D, Raucci, G., Celano, B., Pacilli, A., Zanoni, L., Canevari, S., Adobati, E., Colnaghi, M. I., Dosio, F., Arpicco, S., Cattel, L., Mele, A., and De Santis, R. (1996) Clavin, a type-1 ribosome-inactivating protein fromAspergillus clavatus IFO 8605.Eur. J. Biochem. 239, 272–280.PubMedCrossRefGoogle Scholar
- 8.Gasset, M., Oñaderra, M., Thomas, P. G., and Gavilanes, J. G. (1990) Fusion of phospholipid vescicles produced by the anti-tumor protein α-sarcin.Biochem. J. 265, 818–822.Google Scholar
- 9.Wawrzynczak, E. J., Henry, R. V., Cumber, A. J., Parnell, G. D., Derbyshire, E. J., and Ulbrich, N. (1991) Biochemical, cytotoxic and pharmacokinetic properties of an immunotoxin composed of a mouse monoclonal antibody Fib 75 and the ribosome-inactivating protein α-sarcin fromAspergillus giganteus.Eur. J. Biochem. 196, 203–209.PubMedCrossRefGoogle Scholar
- 13.Lacadena, J., Martinez del Pozo, A., Barbero, J. L., Mancheño, J. M., Gasset, M., Oñaderra, M., Lopez-Otin, C., Ortega, S., Garcia, J., and Gavilanes, J. G. (1994) Overproduction and purification of biologically active native fungal α-sarcin inEscherichia coli.Gene 142, 147–151.PubMedCrossRefGoogle Scholar
- 16.Sambrook, J., Fritsch, E. F., and Maniatis T. (1989)Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar