Cell-free protein biosynthesis - direct ultrahigh sensitive
non-radioactive control.
The new Lumi-Pro kit from the MolTech GmbH, Berlin, Germany, is designed
to
allow continuous control of activity of cell-free lysates during the
translation of m-RNA and control of protein biosynthesis.
The common methods of control, such as radioactive labelling of the
growing
protein chain, measurernent of an enzymatic activity of synthesised
protein,
or bioluminescence of luciferase/luciferin, provide only information about
integral quantity of synthesised protein. Therefore is it difficult to
control
the changes of translation activity of the ribosomal system depending on
the
time or on the addition of some components, like energy sources,
inhibitors,
ions, etc. In the case of radioactive labelling, the shorter, as a full
protein, sequences will also be registered.
We have developed the kit and protocols for continuous registration of the
translational efficiency of different cell-free lysates on the basis of
Ca++-
triggered bioluminescent photoprotein obelin (aequorin-typ) from Obelia
longissima. This
method is also useful in controlling the synthesis of a chimeric
constructions with obelin insertion on ribosorne. Potential applications
of
this method include the screening of drugs, chemicals.antibiotics. The
method
is based on the rapid binding, in the presence of oxygen, of 1 molecule
coelenterazine and 3 Ca++ ions with every obelin-molecule after its
synthesis
and emission of a single photon per each completely synthesised obelin
chain.
Registration of the obelin bioluminescence in the protein biosynthesis
system
is possible by using standard scintillation counter in the single photon
counting modus. The output the "single photon-CPM" is proportional to the
quantity of obelin-molecules synthesised per minute. The integrated value
of
the SP-CPM within 6 hours is approx. 10,000 times higher than the value of
the
radioactivity-CPM in the same protein biosynthesis system.
The kit includes obelin - m-RNA, obelin-cofactor coelenterazine, obelin as
positive control, and the necessary protocols. Those quantities of
reagents
are sufficient for 100 experiments with 25 mcl of cell-free lysate.
The optimised wheat germ lysate, obelin, apo-obelin and coelenterazine are
also available.
Please contact us:
E-Mail: mtberlin at aol.com or contact at pharma-net.com
Fax: +4930/787963-19
Tel: +4930/787963-0
Dr. Natalia Berestovskaia
Molecular Technology GmbH
Czeminskistrasse 1a
10829-Berlin, Germany
