Minuth, T. and Kramer, B. and Lehle, K. and Jaenicke, R. and Kohnert, U. (1998) The spectroscopic analysis, inhibition and binding studies demonstrate the equivalence of Erythrina caffra trypsin inhibitor and the recombinant substitution variant recSerETI. JOURNAL OF BIOTECHNOLOGY, 62 (3). pp. 231-239. ISSN 0168-1656, 1873-4863
Full text not available from this repository.Abstract
A recombinant substitution mutant (recSerETI) of the Erythrina caffi a trypsin inhibitor, with the N-terminal valine residue substituted by serine, was produced in E. coli and compared to the wildtype protein (wtETI) with respect to physicochemical and functional properties. The spectral properties, including UV absorbance, fluorescence emission and circular dichroism, were indistinguishable. Furthermore, the inhibitory activities of the two proteins regarding the inhibition of trypsin, chymotrypsin, tissue plasmininogen activator (t-PA) and reteplase (BM 06.022, t-PA deletion variant comprising the kringle 2 and the protease domains, isolated from transformed E. coli cells) and the affinity of the immobilized inhibitors for reteplase were closely similar. Five repetitive cycles of guanidinium chloride (GdmCl) -induced denaturation-renaturation yield the native mutant protein with its inhibitory activity fully restored. The only difference between the wildtype and the mutant protein refers to the intrinsic stability. Comparing the pH- and GdmCl-dependent transitions, as well as the thermal denaturation, recSerETI exhibits decreased stability compared to the wildtype protein. The pH range of stability is shifted from pH 1-9.5, for wtETI, to pH 2-9, for recSerETI; similarly the GdmCl-induced denaturation is found to occur at a GdmCl half concentration of 3.7 M instead of 4.5 M; in both cases the renaturation exhibits strong hysteresis. The mid-point of the thermal unfolding transition of the mutant protein is at similar to 65 degrees C, as compared to similar to 75 degrees C for the wildtype protein. (C) 1998 Elsevier Science B.V. All rights reserved.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | TISSUE-PLASMINOGEN-ACTIVATOR; SITE-DIRECTED MUTAGENESIS; PROTEASE INHIBITOR; ESCHERICHIA-COLI; DISULFIDE BONDS; PURIFICATION; EXPRESSION; SEEDS; GENE; Erythrina caffra; recSerETI; spectroscopic analysis |
| Subjects: | 500 Science > 540 Chemistry & allied sciences 500 Science > 570 Life sciences |
| Divisions: | Biology, Preclinical Medicine > Institut für Biophysik und physikalische Biochemie |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 23 Feb 2023 10:29 |
| Last Modified: | 23 Feb 2023 10:29 |
| URI: | https://pred.uni-regensburg.de/id/eprint/49668 |
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