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EP0927247A1 - Extrait traductionnel exempt de cellules et obtenu de cellules eukaryotes ayant proliferees en monocouches - Google Patents

Extrait traductionnel exempt de cellules et obtenu de cellules eukaryotes ayant proliferees en monocouches

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Publication number
EP0927247A1
EP0927247A1 EP97939922A EP97939922A EP0927247A1 EP 0927247 A1 EP0927247 A1 EP 0927247A1 EP 97939922 A EP97939922 A EP 97939922A EP 97939922 A EP97939922 A EP 97939922A EP 0927247 A1 EP0927247 A1 EP 0927247A1
Authority
EP
European Patent Office
Prior art keywords
extract
cells
translation
buffer
rnas
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Withdrawn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
EP97939922A
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German (de)
English (en)
Inventor
Daniel Favre
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Individual
Original Assignee
Individual
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Filing date
Publication date
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Publication of EP0927247A1 publication Critical patent/EP0927247A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention relates to a method for generating a cytoplasmic extract from eukaryotic cells that have grown as monolayers and that is efficient in translating endogenous as well as exogenously added ribonucleic acids (RNAs).
  • RNAs ribonucleic acids
  • mRNAs messenger RNAs
  • mRNAs messenger RNAs
  • reticulocytes are not representative of eukaryotic cells in the way they regulate translation.
  • the cells can be: i) preincubated with hormones, toxines, ions, etc. ; or ii) pretreated with chemical or other external stimuli before the generation of the translational extract.
  • the extract can be prepared by employing inexpensive compounds that are available from commercial sources.
  • the extract can be frozen and subsequently thawed for further use.
  • RNAs ribonucleic acids
  • the method may further comprise a step wherein the cells of step a) are washed with a washing buffer having an osmolarity between 0.1 and 500 mOsM.
  • the washing buffer may contain a sugar.
  • the method may further comprise a step wherein the cellular extract of step c) are frozen and thawed for usage.
  • the method may further comprise a step wherein the cells of step b) are centrifuged to collect the supernatant cytoplasmic extract.
  • the extraction buffer include any compounds required for efficient translation of RNAs and may be selected from buffers preserving physiological pH, such as Hepes .
  • the extraction buffer include a transcription enzyme and ribonucleotides for transcription of RNAs from exogenously added DNA, wherein the transcripted RNAs are translated.
  • the preferred extraction buffer includes Hepes- KOH; one salt selected from the group consisting of NH4CI, KCl and potassium acetate; magnesium acetate; ATP; GTP; creatine phosphate; creatine phosphokinase; and each essential amino acid with at least one essential amino acid being radioactively labeled.
  • the extraction buffer may optionally include dithiotreitol; spermidine; or spermine.
  • Figs. 1, 2 and 4 illustrate each a fluorography of radiolabeled polypeptides synthesized in in vitro protein synthesis in accordance with the method of the present invention using BHK cells;
  • Fig. 3 illustrates a fluorography of radiolabeled polypeptides synthesized in in vitro protein synthesis in accordance with the method of the present invention using Rat 6 cells;
  • Fig. 5 illustrates the different enzyme addition protocol for maximum energy generation in accordance with the present method.
  • Eukaryotic cells are grown as monolayers in Petri dishes (for example diameter, 100 mm) in their optimal cell culture medium.
  • BHK cells can be grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with glutamine and 10% inactivated (30 rain., 56°C) fetal calf serum (FCS) or fetal bovine serum (FBS), or newborn calf serum (NBCS) or any other suitable serum.
  • DMEM Dulbecco's Modified Eagle Medium
  • FCS fetal calf serum
  • FBS fetal bovine serum
  • NBCS newborn calf serum
  • cell monolayers (about 10 cells in the case of BHK cells;
  • permeabilization is performed) with 100 ⁇ g/ml lysolecithin (L-lysophosphatidylcholine, palmitoyl; Sigma) in washing buffer. This concentration of lysolecithin might be the minimum concentration that renders the cells permeable to the dye trypan blue.
  • the solution is aspirated and the dishes are drained upright in order to aspirate the residual liquid. The cells are then scraped into 200 ⁇ l (Caeiro, F. and J. V.
  • the translation extract referred as cytoplasmic extract, is obtained after centrifugation of the nuclei at 1500 x g for 5 min. at 4°C using a table centrifuge. These conditions of disruption through a needle and centrifugation are indicative and can be performed by varying the parameters .
  • the translation extract is incubated at 20°C (referred as "room” temperature) for 10 min. (for example, as usually indicated in the scientific literature) in presence of 10 units of micrococcal nuclease (Cuatrecasas, P. et al., 1967, J. Biol . Chem. 242:1541- 1547) (P-L Biotechnology) per ml and 1 mM CaCl2 to hydrolyse the endogenous mRNAs. The reaction is stopped with the addition of 2.5 mM ethylene glycol-bis( ⁇ - aminoethyl ether )-N,N,N' ,N' -tetraacetic acid [pH 7] (EGTA).
  • EGTA ethylene glycol-bis( ⁇ - aminoethyl ether )-N,N,N' ,N' -tetraacetic acid
  • the volume of micrococcal nuclease plus CaCl2 plus EGTA that was added represented, in this case, 4% of the final volume of the cytoplasmic extract.
  • Other concentrations of these latter compounds might also be employed (i.e.: more or less concentrated).
  • Translation reaction (in 40 ⁇ l ) contained 30 ⁇ l of cytoplasmic extract, 0.5 ⁇ Ci/ ⁇ l of [ 35 S]methionine
  • RNAs can be from various sources, for example: a) from biological sources (Chirgwin, J. M. et al., 1979, Biochemistry 18:5294-5299); or b) transcribed in vi tro with or without a 5 '-cap. The reaction is stopped with the addition of 2 x SDS-sample buffer followed by boiling for 3 min.; analysis of the polypeptides by SDS-PAGE was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
  • the cells were grown in monolayers, and disruption of the plasma membrane with lysolecithin was optimized for each cell type as described (Caeiro, F. and J. V. Costa, 1989, Virology 173:728-732). For example, incubation of the cells at 4°C for 90 seconds in washing buffer containing 100 ⁇ g of lysolecithine per ml is performed when BHK cells are employed.
  • GTP for example ranging between 0.05 and 2 mM
  • creatine phosphate up to 40 ⁇ M
  • polyamines for example, from calf liver and at 200 ⁇ g/ml
  • Potassium acetate in the extraction buffer was employed instead of NH4CI or KC1 to avoid the inhibition of initiation of translation that might result from the high concentration of Cl ⁇ ions in the translation reaction (Weber, L. A. et al . , 1977, J. Biol . Chem. 252:4007-4010).
  • NH4CI or KC1 might be employed instead of potassium acetate, if desired.
  • the optimal concentration of Mg 2+ and K + ions are particularly dependent on the nature of the mRNA being translated and ranges between 1.5 to 2.5 mM and between 75 to 110 mM (when KC1 is used; or up to 150 mM when potassium acetate is employed), respectively (Clemens, M.
  • Preincubation of the cells for times ranging between 10 min. to 2 h. in medium lacking methionine does not notably induce a stress response. In fact, a preincubation of about 15 to 30 min. is sufficient and allowed very high incorporation of [ ⁇ Sjmet into newly synthesized polypeptides.
  • depletion of amino acid(s) can be performed for shorter or longer periods of time, if convenient. Following this depletion, it has been mentioned that the petri dishes are incubated on ice for 10 min.; this period of time is indicative and can also be changed, if convenient. It has been suggested that 200 ⁇ l of extraction buffer should be applied to cells growing on a 10 cm diameter petri dish to obtain the above-mentioned cytoplasmic extract.
  • the volume of the extraction buffer required to obtain the cytoplasmic extract must thus be adapted to each case, bearing in mind the surface of the support.
  • 800 ⁇ l of extraction buffer should be employed with a 20 cm diameter petri dish.
  • the cytoplasmic extract (30 ⁇ l ) represented 75% of the final volume of the translational reaction (40 ⁇ l).
  • the final volume of the translational reaction can be changed by any factor of proportionality.
  • the reaction can be performed in 100 ⁇ l by employing 75 ⁇ l of cytoplasmic extract.
  • the volume of the cytoplasmic extract versus the final volume of the translational reaction can be also changed. If this condition is to be achieved, the components of the cytoplasmic extract must thus be adapted by multiplying them with an accurate factor of multiplication, in order to obtain a final concentration of these components which allows efficient in vi tro protein synthesis.
  • the translation reaction can be performed in a final volume of 40 ⁇ l by employing 15 ⁇ l of cytoplasmic extract; in this case, the concentration of the components that are present in the extraction buffer described above must be doubled.
  • glycerol prevents inactivation of enzymes by freeze/thawing.
  • creatine kinase might be added in the final translation extract and the translation extract might be thawed in presence of added glycerol at a convenient final concentration, for example, 5% (vol. /vol.) glycerol. This can be true for pyruvate kinase, too.
  • K -acetate and/or Mg -acetate can be mixed with fresh buffer at convenient ratio; this buffer might thus contain K -acetate and/or Mg acetate and biologically active creatine kinase.
  • Final t ++ concentration of K -acetate/and/or Mg -acetate can thus be adapted accordingly to each translation reaction.
  • cells After permeabilization of the cells, cells can be collected in minimal buffer consisting in Hepes-KOH [pH 7.4] and the cytoplasmic extract can then be generated as indicated above.
  • the cytoplasmic extract freshly made or thawed
  • the cytoplasmic extract must be supplied with translation mix allowing the generation of the translational extract.
  • all the components of the translation mix such as ATP, GTP, K-acetate or KC1, Mg-acetate, etc, can be individually or simultaneously adapted in order to obtain a translational reaction with all the desired concentrations of ATP, GTP, K- acetate or KC1, Mg-acetate (for example).
  • the above list of these compounds is meant to be taken as a non- exhaustive list and the present procedure may be carried out with other compounds .
  • the translation extract (described above) is efficient for translation of exogenous mRNAs. However, it might be that in certain circumstances, extracts could be resistant to translation of exogenously added mRNAs. To overcome this potential inhibition, the following modifications could be employed, in order to check among various possibilities which could explain the block imposed on the initiation of translation of exogenous mRNAs.
  • HRI heme-regulated eIF-2 ⁇ kinase
  • the driving force for polypeptide chain elongation should be an increasing function of the ratios of GTP to GDP and aminoacyl-tRNA to RNA (Kurland, C. G., 1982, Cell 28:201-202). Inhibitory small molecules such as GDP may accumulate during incubation of mammalian cell-free systems, and this may lead to early failure of initiation (Clemens, M. J., 1979, Transcription and translation : a practical approach (B. D. Hames & S. J. Higgins) . IRL Press pp. 231-270). Thus, translations could be performed with the use of, for example, either lower (0.05 mM) or higher (2 mM) concentrations of GTP.
  • Micrococcal nuclease could have a deleterious effect on endogenous tRNA.
  • the addition of, for example, calf liver tRNA up to 200 ⁇ g per ml (for example) in the translation reaction can be performed after the inhibition of the micrococcal nuclease with EGTA or pTp (for pTp, see below).
  • the compound 2 '-deoxythymidine,3 *-5'- diphosphate (pTp; Sigma) is also useful for the inhibition of the micrococcal nuclease (Skup, D. et al., 1977, Nucleic Acids Res . 4:3581-3587).
  • pTp micrococcal nuclease
  • it can be added in place of EGTA to the translation extract to inhibit this enzyme (for example, 0.1 mM pTp instead of 2.5 mM EGTA).
  • the cells can be depleted of (an) amino acid(s) other than methionine (as described above) by preincubating it with medium lacking (an) other amino acid(s).
  • the cells can be preincubated in medium lacking cysteine; the extraction buffer will thus contain all amino acids except cysteine, and the translation reaction will be performed with
  • the translational extract can directly be stored in liquid nitrogen after its preparation, and thawed for a further use. If convenient, it might also be possible to add dimethyl sulfoxide (DMSO) or glycerol to the cytoplasmic extract prior to its freezing. For example, use of up to 4% (vol/vol) of DMSO and 5% glycerol in the translational extract has no deleterious effect on translational efficiency after thawing of the extract.
  • DMSO dimethyl sulfoxide
  • glycerol glycerol
  • the translational extract could also be dialyzed or passed over SEPHADEXTM (coarse) at 4°C to lower the concentrations of amino acids and to standardize the ionic conditions before storage.
  • SEPHADEXTM coarse
  • transcription is dependent of exogenous added deoxyribonucleic acid (DNA) templates.
  • DNA deoxyribonucleic acid
  • This transcription can be performed by using S-adenosyl- methionine as a donor of methyl groups. S-adenosyl- methionine does not interfere with in vi tro translation when added to the extraction buffer that is only employed for translation.
  • Cytoplasmic extract was prepared according to the procedure described above. Extract was treated with micrococcal nuclease (m.n.; indicated by + where necessary on the fluorography), or not treated with micrococcal nuclease (-).
  • CEL RNA is the CAT-EMC-LUC RNA (column 3); the CAT (chloramphenycol acetyl transferase) protein of about 24 kilodaltons (kDa) is indicated by *, the LUC (luciferase) protein of about 65 kDa is indicated by >.
  • BMV is the brome mozaic virus RNA (column 4).
  • 0.2 ⁇ g of CAT-EMC-LUC RNA was employed; and 0.4 ⁇ g of BMV RNA.
  • Columns 3 and 4 show that the activity of micrococcal nuclease was inhibited after incubation and addition of EGTA, since the exogenous added RNAs have been efficiently translated.
  • Cytoplasmic extract was prepared according to the procedure described above. Extract was treated with micrococcal nuclease (m.n.; indicated by + where necessary on the fluorography), or not treated with micrococcal nuclease (-).
  • a control reaction was performed with a Krebs ascites fluid following established procedures and in presence of CAT-EMC-LUC RNA; the positions of the CAT and LUC polypeptides are indicated (column 1).
  • Cytoplasmic extract was prepared according to the procedure described above. Extract was treated with micrococcal nuclease (m.n.; indicated by + where necessary on the fluorography), or not treated with micrococcal nuclease (-).
  • BMV RNA is efficiently translated (columns 3 and 6; with extract not treated or treated with micrococcal nuclease, respectively).
  • Cytoplasmic extract was prepared according to the procedure described above. Extract was treated with micrococcal nuclease (m.n. ; indicated by t where necessary on the fluorography), or not treated with micrococcal nuclease (-). A control reaction that contained no exogenous added RNA, and that was not treated with micrococcal nuclease, was performed: actin and tubulin polypeptides are indicated by arrows (column 1). Extract treated with micrococcal nuclease and employed without exogenous added RNA shows no radiolabeled polypeptide (column 4).
  • both the CAT and the LUC polypeptides are translated (columns 3 and 6; with extract not treated or treated with micrococcal nuclease, respectively).
  • Viral RNA from picornaviruses are efficiently translated: encephalo yocarditis virus (EMC) RNA (0.2 ⁇ g; columns 2 and 5; with extract not treated or treated with micrococcal nuclease, respectively); poliovirus RNA (0.4 ⁇ g; columns 7; with extract that has been treated with micrococcal nuclease).
  • EMC encephalo yocarditis virus
  • RNAs containing an internal ribosome entry site (IRES)
  • brome mozaic virus (BMV) RNA 4′-(BMV) RNA.
  • Example II is essentially illustrated in Fig. 5. Creatine kinase (EC 2.7.3.2) is added to the cytoplasmic extract that has been freshly prepared, prior to the translation reaction. Usually 1 ⁇ l of creatine kinase (at 48 mg/ml that is resuspended in 50%
  • cytoplasmic extract When a cytoplasmic extract is frozen in presence of creatine kinase (for example), and thawed for further use, the cytoplasmic extract is efficient in translating endogenous RNAs, but fails to initiate translation or exogenous added RNAs.
  • the biologically active enzyme must be added freshly to the cytoplasmic extract.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

L'invention porte sur un procédé d'obtention d'un extrait cytoplasmique de cellules eukaryotes ayant proliféré en monocouches, et s'avérant efficace pour traduire les acides ribonucléiques (ARN) endogènes ou à adjonction exogène.
EP97939922A 1996-09-16 1997-09-15 Extrait traductionnel exempt de cellules et obtenu de cellules eukaryotes ayant proliferees en monocouches Withdrawn EP0927247A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US2638496P 1996-09-16 1996-09-16
US26384 1996-09-16
PCT/CA1997/000669 WO1998012315A1 (fr) 1996-09-16 1997-09-15 Extrait traductionnel exempt de cellules et obtenu de cellules eukaryotes ayant proliferees en monocouches

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EP0927247A1 true EP0927247A1 (fr) 1999-07-07

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EP (1) EP0927247A1 (fr)
AU (1) AU4196897A (fr)
WO (1) WO1998012315A1 (fr)

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Publication number Priority date Publication date Assignee Title
EP0259739A1 (fr) * 1986-09-10 1988-03-16 Rhone-Poulenc Inc. Stabilité de cultures lyophilisées

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9812315A1 *

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AU4196897A (en) 1998-04-14
WO1998012315A1 (fr) 1998-03-26

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