| Autor: |
Chao, Q.M., Casalongue, C., Quinn, J.M., Etzler, M.E. |
| Zdroj: |
Archives of Biochemistry and Biophysics; September 1994, Vol. 313 Issue: 2 p346-350, 5p |
| Abstrakt: |
The seed lectin from the legume, Dolichos biflorus, was expressed in Escherichia coliusing the pET expression vector. Replacement of the 22-amino acid signal sequence of this lectin with a methionine increased the level of lectin expression greater than 100-fold. Approximately 20% of the expressed seed lectin was soluble; the remainder was solubilized in 8 M urea and renatured by rapid dilution. No difference in physicochemical properties or activity was detected between the soluble and renatured forms. NH2-terminal amino acid analysis and immunoblots, using antibodies that recognize the COOH-terminus of only the nontruncated subunit of the native heteroligomer, established that the expressed lectin has a primary structure equivalent to subunit I of the native seed lectin. The expressed seed lectin is active as evidenced by its ability to bind to blood group A + H substance-Sepharose and to be specifically eluted from this column with N-acetylgalactosamine. However, a comparison of the activity of the expressed lectin with the native seed lectin using a sensitive ELISA showed that the expressed lectin has a slightly lower affinity for blood group A + H substance than the native seed lectin. The expressed lectin also has a lower Mrthan the seed lectin as determined by molecular exclusion chromatography. |
| Databáze: |
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