Epsilon Archive for Student Projects

Abstract

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.