Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1999-04-07
2001-07-03
Prouty, Rebecca E. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C530S350000
Reexamination Certificate
active
06255093
ABSTRACT:
The invention essentially concerns a DNA molecule which codes for a protein with the enzymatic activity of 3-hydroxybutyrate dehydrogenase (E.C.1.1.1.30), recombinant DNA containing the DNA molecule, correspondingly transformed microorganisms, a process for isolating 3-hydroxybutyrate dehydrogenase by culturing a suitable transformed microorganism as well as a method for determining ketone bodies in the presence of the enzyme.
3-Hydroxybutyrate dehydrogenase (3-HBDH) is an enzyme which catalyses the oxidation of hydroxybutyric acid to acetoacetic acid while simultaneously converting NAD
+
into NADH (Bergmeyer, H. U. et al. (1967), Biochem. J. vol. 102, 423-431):
The enzyme is used to detect ketone bodies. Ketone bodies are understood in particular as acetoacetic acid, 3-hydroxybutyric acid and acetone. The formation of ketone bodies is increased when lipolysis is increased e.g. in insulin deficiency (diabetes mellitus; type I diabetics), when the glucagon concentration is increased and in a fasting state. In such cases the physiological concentration of less than 7 mg/dl can increase to more than 10-fold. Over the past years hydroxybutyric acid has proven to be an extremely reliable parameter for monitoring an insulin therapy.
Ketone bodies are metabolites of fat metabolism. They are formed in the liver and are subsequently metabolized especially in the musculature. Ketone bodies are usually detected qualitatively by an interference-prone test for acetone or acetoacetic acid. However, in comparison to acetone or acetoacetic acid, 3-hydroxybutyric acid (3-HB) is a more dominant and reliable indicator for clinical diagnoses. The ratio of 3-HB to acetone or acetoacetic acid is normally 3:1. In keto-acidoses the ratio increases to 6:1 to 12:1. Furthermore a suitable 3-HBDH should enable the development of a quantitative test for 3-hydroxybutyric acid.
It is known that 3-HBDH occurs in a number of eukaryotic organisms as well as in prokaryotes (Churchill, P. et al. (1992), Biochem. vol. 31, 3793-3799; Marks, A. R. et al. (1992), J. Biol. Chem. vol. 267, 15459-15463; Bergmeyer, H. U. et al. (1967), Biochem. J. vol. 102, 423-431). Nowadays various bacteria serve in particular as a source for the isolation of the 3-HBDH enzyme. However, the classical processes for producing the conventional 3-HBDH from certain microorganisms are very time-consuming and costly, which is due, on the one hand, to the weak expression of 3-HBDH and, on the other hand, the fermentation process for suitable microorganisms is poorly reproducible.
Hence the object of the present invention is to provide a recombinant microbial protein with the enzymatic activity of 3-HBDH.
The object is achieved by a DNA molecule comprising a structural gene which codes for a protein with the enzymatic activity of 3-hydroxybutyrate dehydrogenase and is transformed and expressed in a heterologous microorganism by means of a vector.
The DNA molecule or the 3-HBDH structural gene according to the invention is in particular the gene coding for 3-HBDH which can be obtained from various microorganisms such as for example from bacteria of the genera Rhodobacter, Rhodospirillum and Pseudomonas. Species of microorganisms from the genus Rhodospirillaceae, in particular
Rhodobacter sphaeroides
, have proven to be particularly suitable as a source of the DNA molecule according to the invention. In particular a gene coding for 3-HBDH was obtained from
Rhodobacter sphaeroides
(BMTU 109, DSM 12077) which has a nucleotide sequence according to SEQ ID NO.1 or a degenerate sequence based on the genetic code. The Rhodobacter 3-HBDH structural gene has a homology to corresponding eukaryotic genes of only 46 or 47% and is also characterized by a GC content of ca 68 mol % and the codon TCG preferably coding for the amino acid serine (12 out of a total of 15) and CTG/C coding exclusively for the amino acid leucine (a total of 16). The strain
Rhodobacter sphaeroides
with the internal name BMTU 109 was deposited on the 26.03.1998 at the “Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM)”, Mascheroder Weg 1b, D-38124 Braunschweig under DSM 12077.
According to the invention it is also preferred that the 3-HBDH protein is expressed by using a partial sequence contained in SEQ ID NO.1 which is inserted into a suitable vector. Those DNA molecules have proven to be particularly suitable according to the invention which correspond to a nucleotide sequence from nucleotide number 467 to number 1237 of the sequence according to SEQ ID NO.1, which are complementary to this and/or hybridize with this. Such DNA molecules include for example fragments, modifications, derivatives and all variants of the DNA molecules described above. In this connection the term “hybridization” means a hybridization under conventional hybridization conditions, preferably under stringent conditions.
Examples of stringent conditions suitable for use in accordance with the invention can be found in Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, New York, which is hereby incorporated by reference. See specifically sections 1.101-1.104 and 9.47-9.55, which provide conditions well known to those of skill in the art. For example, suitable stringent conditions include washing for one hour in the presence of 1×SSC and 0.1% SDS at 55° C., preferably at 62° C. A further preferred method is using 0.2×SSC and 0.1% SDS at 55° C. The hybridization conditions mentioned in this reference are in general suitable in order to determine DNA molecules which are complementary to and/or hybridize with the nucleic acid sequences in accordance with the invention.
It should additionally be emphasized that in principle several parameters must be considered when calculating the optimal stringent hybridization conditions including, e.g., the probe length, probe concentration, pH value, temperature, and the presence of monovalent cations and/or formamide.
The following specific stringent hybridization conditions have been successfully applied in the present invention: 50% formamide, 5×SSC, 2% blocking reagent (for decrease of background; Roche Molecular Biochemicals, Cat. No. 1 096 176), 1% lauroylsarcosin and 0.02% SDS at 42° C., with a hybridization time of 1-16 hours.
A further subject matter of the invention is a process for the production of a protein with 3-HBDH activity by inserting a DNA molecule coding for 3-HBDH into a suitable vector and transforming the recombinant DNA obtained into a microorganism which is able to produce 3-HBDH in the medium. Suitable microorganisms for the expression are in particular those of the genus
E. coli
such as for example HB101 (ATCC 33694), JM83 (ATCC 35607) and RR1 (ATCC 35102).
According to the invention all plasmid vectors have proven to be suitable for such a process which are usually used for expression in host cells. Vectors based on the plasmid pKK177 under the control of a T5 promoter are mentioned here as an example. After fermentation of the transformed host cells for several hours, the accumulated protein with the enzymatic activity of 3-HBDH is isolated from the culture medium. The enzyme is essentially isolated by methods known to a person skilled in the art such as by centrifugation, lysis of the biomass, resuspension in a suitable buffer solution, separation of insoluble cell components and by various chromatographic steps for example by means of hydrophobic column materials (e.g. phenyl Sepharose) or/and suitable affinity chromatography.
The recombinant 3-HBDH enzyme can be obtained from the heterologous microorganism in good yields with a purity of ca. 60% to 80% and with a specific activity of at least 200 U/mg. The recombinant bacterial enzyme according to the invention is shortened at the C-terminal end compared to the known eukaryotic 3-HBDH enzymes by preferably ca. 30 amino acids and additionally has no phosphatidylcholine dependency. An advantage of the latter is that it is not necessary to add external lipid (phosphatidylcholine) to develop the e
Engel Alfred
Krüger Kerstin
Muller Rainer
Schmuck Rainer
Weisser Harald
Arent Fox Plotkin Kintner Kahn PLLC.
Monshipouri M.
Prouty Rebecca E.
Roche Diagnostics GmbH
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