DE4432053A1 - Use of Listeria bacteriophage coded lysine - Google Patents

Abstract

Listeria bacteriophage coded lysine is used to solubilise bacterial cell suspensions of the Listeria genus, by adding a small amt. of the purified lysine prepn. to the cell suspension for effective prepn. and purificn. of cellular components such as chromosomal DNA, plasmids, RNA and proteins.

Description

Field of the Invention

To examine the cellular components of Listeria (e.g. chromosomal DNA, plasmids, RNA, proteins, lipids, metabolites) must Cells are opened (destroyed) as quickly and gently as possible can to the integrity and functionality of the isolated components and to ensure macromolecules.

State of the art

So far there are 4 methods for obtaining nucleic acids or proteins Listeria cells available: (1) The enzymatic destruction of the cell wall by lysozyme (magazine "Infection and Immunity", 1984, year 44, p. 157-161) is very slow and inefficient, therefore not suitable for the Representation of m-RNA and cell wall-associated proteins. (2) The Combination of enzymatic and chemical destruction of the cell wall (Journal "Letter. In Applied Microbiology", 1989, Volume 8, pp. 151-156; Journal of Bacteriology, 1994, year 176, pp. 3040-3048) is also not very efficient and is still quite complex to use Execution. In addition, proteins are irreversibly denatured, which is a major disadvantage is. (3) The mechanical-physical cell disruption using ultrasound (journal "Medical Microbiology and Immunology", 1992, year 181, pages 283-291) or (4) relief from overpressure (e.g. "French-Press System") only enables the less productive one Isolation of cellular proteins while nucleic acids are being destroyed and Proteins that are bound to the cell wall cannot be released.

The method described below does not have the disadvantages mentioned is very easy and quick to carry out, and is suitable for efficient isolation of all nucleic acids and proteins.  

Description of the invention

So far, numerous bacteriophages have been isolated for the genus Listeria and described, including many of their own isolates from this laboratory. As final product of the expression of the so-called "late genes" during the Phage infection a cell wall lytic enzyme (phage lysine) is formed, which enables the release of the newly formed viruses from the cells. These lysines specifically cleave the peptide bonds between them Murein layers of the Listeria cell walls (L-alanine amidases, D-glutamine Amidases).

By externally adding purified lysine preparations from the Listeriaphagen A511 or A118 for cell suspensions from Listeria becomes one rapid (2-6 min.) and complete (95-100%) cell wall hydrolysis achieved. In addition to components inside the cell (chromosomal DNA, plasmids, RNA, proteins, etc.) also all cell wall-associated (non-covalent bound) proteins released. All macromolecules are then lying native, active and not exposed to chemical damage. Trials in this laboratory showed approximately 50-80% better efficiency in Regarding the isolation of nucleic acids, in addition to a time saving of several hours per approach.

This method and its molecular biological basis was still published anywhere.

Production and storage of the lysines

The biochemical isolation of the above Phage lysine was up to purity carried out. In addition to purifying the enzymes from phage lysates the lysines were obtained from recombinant strains of Escherichia coli, which the corresponding genes from Listeriaphagen A511 or A118 carry an expression plasmid. Here, the cells can be high Yield (up to 20% of the total protein) produce lysines. These recombinant enzymes are easier to isolate and purify, and have the same specificity with no detectable external activity. The purified amidases are in an aqueous buffer system at depth Temperatures (-30 ° C) are kept and are stable for at least 6 months.

Commercial use

Production, cleaning and sale of Listeriaphagen-Lysin preparations Research institutes or similar. There is no comparable alternative for this  Method of fast and gentle cell disruption.

Embodiment

The representation of the cellular expression status at the transcription level demands the quickest possible and gentle isolation of the current in mRNA molecules present in the cell, some of which are extremely unstable.

For the isolation of m-RNA from cultures of Listeria inonocytogenes cells in small volumes (approx. 1-5 ml) by centrifugation concentrated, briefly frozen in a dry ice-ethanol mixture (approx. 1-5 min.), and in 1/10 volume of a suitable buffer (e.g. Tris-HCl, 20 mM, pH 8.0) resuspended. After adding a small amount of the recombinant Lysine (A118 amidase, in 20mM Tris-HCl, pH 7.3, 20mM NaCl, 0.1% Triton X100) is incubated for about 2-5 minutes at room temperature until the cloudy Suspension has become almost completely clear. After deproteination of the Suspension (with phenol: chloroform: isoamyl alcohol, 125: 24: 1 (v / v), pH 4.2) the RNA can be obtained directly by ethanolic precipitation. Remnants of DNA can optionally be removed by hydrolytic cleavage with DNAse will. The now completely pure RNA is available for further examinations Available.

Claims (2)

Use of Listeria bacteriophage-encoded lysines for the digestion of bacterial suspensions of the genus Listeria, with the aim of the efficient and gentle presentation and purification of cellular components such as chromosomal DNA, plasmids, RNA and proteins. The method is characterized in that small amounts of purified lysine preparations are added to the cell suspension. Production, purification and marketing of the lysine. The production happens with recombinant strains of Escherichia coli K-12, which carry the gene (s) for the lysines on an expression plasmid.