Optimization of CarG crystals
Carbapenems are one of the most widely used groups of antibiotics because of their broad spectrum against both gram positive and gram negative bacteria. The biosynthesis of the simplest carbapenem, 1-carbapen-2-em-3- carboxylic acid (Car) is controlled by an operon of eight genes, CarA-H. CarG i...
| Autor principal: | |
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| Formato: | First cycle, G2E |
| Lenguaje: | sueco Inglés |
| Publicado: |
2012
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| Acceso en línea: | https://stud.epsilon.slu.se/4331/ |
| _version_ | 1855570666398416896 |
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| author | Persson, Teres |
| author_browse | Persson, Teres |
| author_facet | Persson, Teres |
| author_sort | Persson, Teres |
| collection | Epsilon Archive for Student Projects |
| description | Carbapenems are one of the most widely used groups of antibiotics because
of their broad spectrum against both gram positive and gram negative
bacteria. The biosynthesis of the simplest carbapenem, 1-carbapen-2-em-3-
carboxylic acid (Car) is controlled by an operon of eight genes, CarA-H.
CarG is believed to possess a function of self-resistance but the mechanism
is not solved. The structure of CarG was solved a few years ago. The aim of
this study was to improve the crystal quality to open up for further binding
studies on CarG.
The protein was purified in a 4 step process; Ni2+-column, desalting,
MonoQ and gelfiltration. MonoQ was added to the previous protocol with
hope to further purify the protein. Different screening protocols were used
and as in earlier studies of CarG, AmSO4 was a suitable precipitant. Crystals
were obtained in a concentration range between 1.3-1.8 M AmSO4 and pH
7.5 and 8, respectively. Using these conditions seeding techniques and
cocrystallization with imipenem and meropenem were performed.
In this study I show that by adding MonoQ chromatography to the
purification protocol bigger crystals and more single crystals can be
obtained. Seeding, in drops with no substrate added, resulted in more
square-shaped crystals but often much thinner crystals. Results from
cocrystallization and seeding with imipenem show no improvement in
crystal quality. Cocrystallization with meropenem gave clear crystals but
was not further improved by seeding. |
| format | First cycle, G2E |
| id | RepoSLU4331 |
| institution | Swedish University of Agricultural Sciences |
| language | Swedish Inglés |
| publishDate | 2012 |
| publishDateSort | 2012 |
| record_format | eprints |
| spelling | RepoSLU43312012-06-28T12:37:11Z https://stud.epsilon.slu.se/4331/ Optimization of CarG crystals Persson, Teres Chemistry Carbapenems are one of the most widely used groups of antibiotics because of their broad spectrum against both gram positive and gram negative bacteria. The biosynthesis of the simplest carbapenem, 1-carbapen-2-em-3- carboxylic acid (Car) is controlled by an operon of eight genes, CarA-H. CarG is believed to possess a function of self-resistance but the mechanism is not solved. The structure of CarG was solved a few years ago. The aim of this study was to improve the crystal quality to open up for further binding studies on CarG. The protein was purified in a 4 step process; Ni2+-column, desalting, MonoQ and gelfiltration. MonoQ was added to the previous protocol with hope to further purify the protein. Different screening protocols were used and as in earlier studies of CarG, AmSO4 was a suitable precipitant. Crystals were obtained in a concentration range between 1.3-1.8 M AmSO4 and pH 7.5 and 8, respectively. Using these conditions seeding techniques and cocrystallization with imipenem and meropenem were performed. In this study I show that by adding MonoQ chromatography to the purification protocol bigger crystals and more single crystals can be obtained. Seeding, in drops with no substrate added, resulted in more square-shaped crystals but often much thinner crystals. Results from cocrystallization and seeding with imipenem show no improvement in crystal quality. Cocrystallization with meropenem gave clear crystals but was not further improved by seeding. 2012-06-20 First cycle, G2E NonPeerReviewed application/pdf sv https://stud.epsilon.slu.se/4331/7/persson_t_120620.pdf Persson, Teres, 2012. Optimization of CarG crystals : a protein involved in the biosynthesis of 1-carbapen-2-em-3-carboxylic acid (Car); a beta-lactam antibiotic. First cycle, G2E. Uppsala: (NL, NJ) > Dept. of Molecular Biology (until 131231) <https://stud.epsilon.slu.se/view/divisions/4025.html> urn:nbn:se:slu:epsilon-s-1285 eng |
| spellingShingle | Chemistry Persson, Teres Optimization of CarG crystals |
| title | Optimization of CarG crystals
|
| title_full | Optimization of CarG crystals
|
| title_fullStr | Optimization of CarG crystals
|
| title_full_unstemmed | Optimization of CarG crystals
|
| title_short | Optimization of CarG crystals
|
| title_sort | optimization of carg crystals |
| topic | Chemistry |
| url | https://stud.epsilon.slu.se/4331/ https://stud.epsilon.slu.se/4331/ |