Method for labeling biopolymers using isotopes

#The present invention concerns a new method for in vivo labeling of biopolymers, like proteins, nucleic acids, lipids, carbohydrates, and biodegradable plastic, with isotopes, especially stable isotopes and the use of chemolithotrophic bacteria for in vivo isotope labeling of biopolymers. The invention concerns in particular the use of CO2-fixing bacteria, like Ralstonia eutropha and similar methanogenic bacteria for in vivo isotope labeling, especially for labeling with the stable isotope 3C and the use of isotope-labeled biomolecules in therapeutic and diagnostic applications, especially in spectroscopy methods and generally as tracer compounds.

#The deliberate use of biomolecules in diagnosis and therapy requires knowledge of the structure and dynamics of these molecules. This information can be obtained, for example, by using nuclear magnetic resonance (NMR). A prerequisite for efficient use of this method, however, is labeling of the biomolecules of interest with stable isotopes, so-called S isotopes, like 2H, 13C and 15N.

#Since S isotopes are not radioactive and, except for deuterium, are not toxic either, they have considerable diagnostic potential. S isotopes have already been successfully used in metabolic diagnosis. In addition to 2H, 13C and 15N, the nuclei 1H, 7Li, 11B, 14N, 17O, 18O, 19F, 23Na, 29Si, 31P, 33S and 77Se have been used above all for NMR spectroscopy of organic compounds (cf., Vogel, H. J. (1989), Methods in Enzymology, Vol. 177, 263).

#For a fuller understanding of the invention, reference is had to the following description taken in connection with the accompanying drawings, in which:

#FIG. 1 is a showing of plasmid pCH591;

#FIG. 2 shows the Hind III/Eco RI fragment of pOF 39 containing GroESL gene and the cloning into plasmid pCH591 of FIG. 1; and

#In contrast to green algae, which require electromagnetic radiation for fixation of carbon dioxide, chemolithotrophic bacteria use chemical redox reactions as energy source in which inorganic substrates are used as hydrogen donor. Carbon dioxide, which is fixed in the Calvin cycle, serves as carbon source for buildup of cell substance. Chemolithotropic bacteria are generally divided into the following subgroups: (i) nitrifying bacteria, in whose energy metabolism ammonium is oxidized to nitrite and then to nitrate or nitric acid, (ii) sulfur bacteria, which produce their metabolic energy by oxidation of one or more differently reduced or partially reduced sulfur compounds, (iii) iron- and manganese-oxidizing bacteria, and (iv) methanogenic bacteria, which include Ralstonia eutropha and the genus Hydrogenomonas which produce the required energy from reaction of oxygen and hydrogen, whose growth therefore requires the presence of hydrogen and oxygen. In contrast to most sulfur and nitrifying bacteria, the so-called methanogenic bacteria are only facultatively chemoautotrophic. This means that they grow heterotrophically in the presence of organic matter, like most bacteria and animals, and switch to the corresponding adequate type of metabolism in adaptation to the prevailing growth conditions. This offers the advantage that genetic manipulations in Ralstonia eutropha and other methanogenic bacteria can be conveniently conducted in the complex medium without H2 and O2.

#The isotope labeling according to the invention is more cost-effective and less demanding than the methods of the prior art. In particular, the method according to the invention is characterized by the use of less costly and less complex growth media. In the case of labeling with C isotopes, for example, the stable 13C isotope, the detour via culturing of green algae on appropriately labelled, for example, 13C-labeled carbon dioxide and production of algal hydrolyzate also drops out.

#Moreover, CO2-fixing bacteria, like Ralstonia eutropha, and other strains can also be used for production of isotope-labeled hydrolyzate in cases in which labeling Escherichia coli is inaccessible, as in green algae and their hydrolyzates.