EP1517986A2 - Decomposition cellulaire effectuee par des bacteries - Google Patents

Decomposition cellulaire effectuee par des bacteries

Info

Publication number
EP1517986A2
EP1517986A2 EP03762592A EP03762592A EP1517986A2 EP 1517986 A2 EP1517986 A2 EP 1517986A2 EP 03762592 A EP03762592 A EP 03762592A EP 03762592 A EP03762592 A EP 03762592A EP 1517986 A2 EP1517986 A2 EP 1517986A2
Authority
EP
European Patent Office
Prior art keywords
amino acids
domain
cells
substances
cytoskeleton
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03762592A
Other languages
German (de)
English (en)
Inventor
Frank Mayer
Andreas Schwienhorst
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novologix GmbH
Original Assignee
Novologix GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novologix GmbH filed Critical Novologix GmbH
Publication of EP1517986A2 publication Critical patent/EP1517986A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms

Definitions

  • the invention relates to the use of substances that bind to the bacterial elongation factor EF-Tu for cell disruption or for the lysis of cells.
  • the invention further relates to a cell disruption system or a cell disruption method.
  • Cells in particular bacterial cells, are frequently used to produce compounds using molecular genetic techniques, for example by homologous or heterologous gene expression and expression of synthetically active enzymes.
  • Peptide compounds, triglycerides, wax esters and PHAs are preferably produced, and in particular those compounds which can be used in biotechnology, medicine, in the pharmaceutical sector or in other fields.
  • heterologous gene expression the compounds formed are non-natural components of the cells or bacteria used.
  • the production of a wide variety of compounds by means of gene expression has been extensively described in the prior art (see, for example, Q. Bi et al., Applied Biochemistry & Biotechnology, 95 (1) (2001) 23-30; D.
  • the production of the compounds produced in the cells generally requires that the cells or bacteria have to be lysed.
  • a Such lysis is always mandatory if the desired compounds cannot be removed from the living cell. In this case, the desired compounds can only be released by lysis and sent for further processing (down-stream processing).
  • enzymes such as lysozyme
  • lysozyme can be added which lyse the cell envelope, whereby lysis could not be achieved by induced lysozyme expression.
  • the cell envelope is destroyed, the cell can be regarded as "unlocked" (TR Hopkins, Bioprocess Technology (New York) 12 (1991), 57-83; JA Asenjo et al., Bioprocess Technology (New York) 9 (1990) 143- 75).
  • Methods can also be used for lysis in which mechanical shear forces come into effect to destroy the cells. An example of such a method is the use of a French press.
  • the object of the present invention was therefore to provide an improved method for cell disruption, in particular a method which can be used advantageously in biotechnological production processes.
  • This object is achieved according to the invention by the use of substances which bind to components of the cytoskeleton, in particular to EF-Tu, for cell disruption.
  • substances which bind to components of the cytoskeleton in particular to EF-Tu, for cell disruption.
  • substances which bind to EF-Tu are preferably used to destabilize the cytoskeleton.
  • the bacterial protein EF-Tu contains domains 1, 2 and 3 (H. Song et al., J. Mol. Biol. 285 (1 999) 1 245-1 256).
  • the sequences of the EF-Tu protein and its coding gene have been published for Escherichia coli and a number of other Eu bacteria and are accessible in databases.
  • EF-Tu is a protein that contains three domains.
  • domain 1 includes amino acids 8 to 204, amino acids 172 to 204 forming a connection structure to domain 2.
  • Domain 2 includes amino acids 205 to 298 and domain 3 includes amino acids 299 to 394.
  • EF-Tu is a 3-domain protein, as shown in Figure 1.
  • the protofilaments of the cytoskeleton arise in the living cell in that domains 2 and 3 of EF-Tu proteins (monomer or integrated in a protofilament and only transiently free) with domains 3 and 2 neighboring EF-Tu proteins interact.
  • domains 2 and 3 of EF-Tu proteins monomer or integrated in a protofilament and only transiently free
  • domains 3 and 2 neighboring EF-Tu proteins interact.
  • chains so-called protofilaments, are formed in this way, which combine to form a network (see figure 2).
  • Other factors may be associated with this network, such as FtsZ, MreB and / or M6I.
  • Stability and / or expression of such protofilaments and networks in bacterial cells can be prevented according to the invention, the death of the bacterial cell occurring as a result of lysis.
  • a particularly advantageous procedure of the invention is as follows.
  • the DNA section of the EF-Tu gene from Escherichia coli, which codes for domain 3, is obtained and transferred and expressed in E. coli cells by means of molecular genetic techniques.
  • the cells still have the native EF-Tu gene and can express it.
  • the additionally synthesized domain 3 polypeptides compete with the native EF-Tu proteins for the binding sites for the formation of the protofilaments.
  • a domain 3 polypeptide is incorporated as a chain link instead of a native EF-Tu protein, this leads to chain termination due to the lack of the second binding site (domain 2 is essential for chain formation) on the domain 3 polypeptide.
  • the protofilament stops growing, the cytoskeletal network is weakened and eventually collapses.
  • the cell's cytoplasmic membrane loses its suspension.
  • the cytoplasmic membrane is suspended from the cytoskeleton. This destroys the cell wall, which is positioned and supported in the living bacterial cell by the cytoplasmic membrane (see FIG. 5). Loss of cell wall and cytoplasmic membrane means an exposure of the cell contents so that the cell is lysed (see FIGS. 5 and 6). The cell content can thus be obtained without further cell disruption.
  • Cells which are used for biotechnological production processes, such as heterologous expression, production of proteins, in particular for biotechnological or medical purposes, can thus be disrupted by introducing a sequence into them prior to their use in production which Species corresponding domain of the EF-Tu coded.
  • the substances used for cell disruption contain fractions which bind to EF-Tu in the region of amino acids 218 to 224 of domain 2 and / or in the region of amino acids 317 to 328 or / and 343 to 354 of domain 3.
  • Different secondary structures occur within domains 2 and 3.
  • intracellularly expressed peptide substances are used which are based on oligopeptides which bind to EF-Tu, preferably in the region of the matching sites of domains 2 or / and 3.
  • oligopeptides can contain sections of the amino acid sequences of domains 2 or / and 3 with a length of preferably 4 to preferably 20 amino acids, particularly preferably 5 to 15 amino acids and particularly preferably with a length of 6 to 12 amino acids.
  • the substances contain partial sections or the total range of the amino acid sequences from domain 3 with a length of at least 4 and in particular at least 5 amino acids and which at the same time does not correspond to any section of the amino acid sequences from domain 2.
  • intracellularly expressed peptidic compounds In addition to intracellularly expressed peptidic compounds, intracellularly expressed peptidomimetics can also be used advantageously.
  • Another object of the invention is a method for disrupting cells, in which components of the cytoskeleton are destabilized in the cells.
  • Cytoskeletal elements that can be altered to weaken the cytoskeleton include, for example, FtsZ, MReB, Mbl, and contractile proteins, and particularly EF-Tu.
  • substances which bind to constituents of the cytoskeleton, in particular to EF-Tu, and which have been explained above, are particularly preferably used.
  • the invention can be used particularly advantageously in a method for producing a compound, for example a protein, by using cells in which this protein is expressed, for example by a biotechnological production process, and by introducing a sequence into these cells beforehand which is suitable for a Coded substance, which destabilizes components of the cytoskeleton of the cells.
  • the lysis usually required in conventional biotechnological process control for obtaining the compounds formed in genetically manipulated bacteria can be carried out here with the cell disruption described here. It is advantageous that the cell disruption can be carried out at a precisely defined point in time. This can be an indicator of this Reaching the desired cell density (quorum sensing). An induction of the lysis by quorum sensing takes place in that the cell culture is controlled in its growth behavior and when it is determined that the desired state of the cell culture has been reached, the gene introduced is expressed.
  • a construct is introduced into the cells which, in addition to the gene for the destabilizing substance, contains further coding sequences which make it possible to induce the start of the synthesis of the substance, for example the domain 3 polypeptide, from the outside. In this way it is possible to precisely control the time of cell lysis in the cell culture from the outside.
  • Induction by a quorum-coupled or / and cell culture-self-sufficient process is another suitable form for initiating cell lysis.
  • the invention further comprises a construct comprising a sequence which codes for a constituent of the substance destabilizing the cytoskeleton of cells.
  • This construct preferably further comprises at least one gene segment which allows the induction of the synthesis of the substance destabilizing the cytoskeleton.
  • FIG. 7 Advantageous designs of the construct are shown in FIG. 7. As soon as the induction has taken place, the lysis of the cells is brought about within a cell generation by the interaction of EF-Tu, domain 3 polypeptide, cytoplasmic membrane and cell wall.
  • Fig. 1 a EF-Tu (GDP form from E. coli; result of an X-ray structure analysis; figure taken from the literature and modified).
  • the 3 domains of the protein molecule are marked.
  • a 'and b denote connection sequences b) Like a), but different form of representation.
  • the numbers mark the positions of the amino acid residues; N-terminus and C-terminus are given.
  • Gold markers used for marking purposes.
  • EF-Tu-GFP-His fusion clones are to be generated.
  • the ideal fusion point for EF-Tu is the C-terminus, which protrudes between domains 2 and 3 (Song et al., 1 999).
  • the same fusion point can also be chosen for a domain-3 fusion construct.
  • the domain 3 fusion construct can be used for in vivo competition experiments. It seems sensible to introduce a C-terminal cysteine (the only one in domain 3) for chemical labeling alternatives.
  • the starting point is genomic DNA from an E. coli K-12 strain. in the
  • Tu was expressed in addition to the cell's own EF-Tu;
  • Lad Repressor p u ac o- ⁇ modified Lac promoter (according to LUTZ and BUJARD, 1 997)
  • the promoters ara, T7 or bdA (quorum sensing) can also be used as alternatives to the specified promoter.
  • Clones are plated on selection agar (here: LB / ampicillin) and grown overnight at 37 ° C
  • I I Inoculate a 1 I LB / ampicillin liquid culture, incubate at 37 ° C up to an OD600 of approx. 0.5 to 0.7, induction with IPTG for at least 4 h

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Medicinal Chemistry (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Mycology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

L'invention concerne l'utilisation de substances se fixant au facteur d'élongation ET-Tu bactérien, pour effectuer la décomposition de cellules ou la lyse de cellules. L'invention concerne en outre un système de décomposition cellulaire et une méthode de décomposition cellulaire.
EP03762592A 2002-07-02 2003-07-02 Decomposition cellulaire effectuee par des bacteries Withdrawn EP1517986A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10229645A DE10229645A1 (de) 2002-07-02 2002-07-02 Zellaufschluss von Bakterien
DE10229645 2002-07-02
PCT/EP2003/007068 WO2004005506A2 (fr) 2002-07-02 2003-07-02 Decomposition cellulaire effectuee par des bacteries

Publications (1)

Publication Number Publication Date
EP1517986A2 true EP1517986A2 (fr) 2005-03-30

Family

ID=30009775

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03762592A Withdrawn EP1517986A2 (fr) 2002-07-02 2003-07-02 Decomposition cellulaire effectuee par des bacteries

Country Status (5)

Country Link
US (1) US20060172407A1 (fr)
EP (1) EP1517986A2 (fr)
AU (1) AU2003249934A1 (fr)
DE (1) DE10229645A1 (fr)
WO (1) WO2004005506A2 (fr)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002087554A2 (fr) * 2001-04-30 2002-11-07 Novologix Gmbh Agent antibacterien

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE19929485B4 (de) * 1999-06-28 2006-07-27 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Lytisches Enzym

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002087554A2 (fr) * 2001-04-30 2002-11-07 Novologix Gmbh Agent antibacterien

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DABORA R.L.; COONEY C.L.: "Intracellular lytic enzyme systems and their use for disruption of Escherichia coli", ADVANCES IN BIOCHEMICAL ENGINEERING, BIOTECHNOLOGY,, vol. 43, 1990, pages 11 - 30, XP009114984, DOI: doi:10.1007/BFb0009077 *
MAYER F.; VOGT B.; POC C.: "Immunoelectron microscopic studies indicate the existence of a cell shape preserving cytoskeleton in prokaryotes.", DIE NATURWISSENSCHAFTEN, vol. 85, no. 6, 1998, pages 278 - 282 *
MAYER FRANK: "Cytoskeletons in prokaryotes.", CELL BIOLOGY INTERNATIONAL, vol. 27, no. 5, 2003, pages 429 - 438 *
MOLLER-JENSEN JAKOB; LÖWE JAN: "Increasing complexity of the bacterial cytoskeleton.", CURRENT OPINION IN CELL BIOLOGY, vol. 17, no. 1, February 2005 (2005-02-01), pages 75 - 81 *
See also references of WO2004005506A3 *

Also Published As

Publication number Publication date
US20060172407A1 (en) 2006-08-03
WO2004005506A2 (fr) 2004-01-15
WO2004005506A3 (fr) 2004-05-06
AU2003249934A1 (en) 2004-01-23
DE10229645A1 (de) 2004-05-19
AU2003249934A8 (en) 2004-01-23

Similar Documents

Publication Publication Date Title
DE69434520T2 (de) Biotinylierung von proteinen
DE68928532T2 (de) Funktionelles, durch ein rekombinantes verfahren hergestelltes synthetisches proteinpolymer
DE69128362T2 (de) Zusammensetzungen und verfahren zur identifizierung von molekülen mit biologischer wirksamkeit
DE3884529T2 (de) Leader-anteile zur produktion von rekombinanten proteinen.
DE69936103T2 (de) Fusionsproteine bestehend aus proteingerüst und bibliotheken von zufälligen peptiden
DE3752363T2 (de) Herstellung synthetischer dns und deren verwendung bei der synthese grosser polypeptide
DE60208343T2 (de) FUSIONSPROTEINE aus hirudin und proinsulin oder insulin
DE69636536T2 (de) Sekretionssignal-Gen und dieses enthaltender Expressionsvektor
DE212012000234U1 (de) Modifzierte Cascade-Ribonukleoproteine und Anwendungen davon
SK116896A3 (en) Enhanced secretion of polypeptides
FI87800C (fi) Foerfarande foer framstaellning av glukagon i jaest
DE112006003608T5 (de) Verfahren zur Präsentation von Zielproteinen an der Zelloberfläche unter Verwendung von Bacillus Anthracis-Exosporium
DE102020205703A1 (de) Expression von Kollagenpeptid-Komponenten in prokaryotischen Systemen
EP0875567B1 (fr) Protéines à doigts de zinc se liant à l'oncogène mycm ainsi comment leur préparation et utilisation
DE69935313T2 (de) Nachweisverfahren für peptide
DE69629808T2 (de) Promotoren für Gen Expression
DE10196565B4 (de) Rekombinantes Human-Parathyroidhormon erzeugender Hefe-Transformant und Verfahren zur Herstellung des Hormons
DE69930938T2 (de) Verfahren zur herstellung eines peptids mit einem pi grösser als 8 oder kleiner als 5
DE69931382T2 (de) Intein vermittelte peptidligation
DE69629460T2 (de) Biologisches verfahren zur herstellung von verbindungen auf der basis der dipeptiden
DE68923496T2 (de) Verfahren zur Herstellung von motilinähnlichen Polypeptiden und seine Expression.
EP1517986A2 (fr) Decomposition cellulaire effectuee par des bacteries
DE69936363T2 (de) DNAs die für neue fusions-Proteine kodieren und Verfahren zur Herstellung von nützlichen Polypeptiden durch Expression der DNAs
EP2513305B1 (fr) Système de sécrétion à base d'une séquence signal d'hydrophobine de trichoderma reesei
DE69603004T2 (de) Temperaturstabile geranylgeranyl disphosphat synthase

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20041222

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20060620

GRAP Despatch of communication of intention to grant a patent

Free format text: ORIGINAL CODE: EPIDOSNIGR1

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20100729