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Mayr, Lorenz M. ; Schmid, Franz X.:
A purification method for labile variants of ribonuclease T1.
In: Protein Expression and Purification.
Bd. 4
(1993)
Heft 1
.
- S. 52-58.
ISSN 1046-5928
DOI: https://doi.org/10.1006/prep.1993.1008
Abstract
We present a new procedure for the rapid production of ribonuclease T1 variants with decreased stability which could not be purified in satisfying amounts by the existing methods. The major changes from the established procedures are the following. (i) The cells were grown at 28 degrees C rather than at 37 degrees C. (ii) The entire purification was performed at low temperatures (4 degrees C). (iii) Materials for chromatography with high flow rates were used to accelerate protein isolation. (iv) The pH was lowered from 7.5 to 6.0, a condition under which RNase T1 is much more stable. The use of this improved procedure allowed the purification of the labile P39G and P73V variants of RNase T1. By the same technique 300 mg of the wild-type protein could be isolated from 10 liters liquid culture within 3 days. The P39G and the P73V mutations strongly decrease the stability of RNase T1 and the midpoints of the reversible thermal unfolding transition are lowered by 16 and 6 degrees C, respectively, relative to that of the wild-type protein. The decrease in temperature during fermentation and the rapid purification at low temperature and under solvent conditions where the stability of the proteins is high are probably the major reasons for the dramatic increase in yield of these labile variants of RNase T1. Such an approach should be valuable for the production of recombinant proteins in general.
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| Publikationsform: | Artikel in einer Zeitschrift |
|---|---|
| Begutachteter Beitrag: | Ja |
| Zusätzliche Informationen: | PubMed-ID: 8425108 |
| Institutionen der Universität: | Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Ehemalige Professoren > Professur Biochemie - Univ.-Prof. Dr. Franz Xaver Schmid Fakultäten Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Professur Biochemie Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Ehemalige Professoren |
| Titel an der UBT entstanden: | Ja |
| Themengebiete aus DDC: | 500 Naturwissenschaften und Mathematik > 540 Chemie |
| Eingestellt am: | 13 Mai 2015 10:29 |
| Letzte Änderung: | 05 Sep 2022 11:47 |
| URI: | https://eref.uni-bayreuth.de/id/eprint/13466 |