WO1998031827A1

WO1998031827A1 - Production of biologically active polypeptides

Info

Publication number: WO1998031827A1
Application number: PCT/IB1998/000078
Authority: WO
Inventors: Daniel Favre
Original assignee: Daniel Favre
Priority date: 1997-01-21
Filing date: 1998-01-20
Publication date: 1998-07-23
Also published as: EP0953055A1; AU5337198A

Abstract

The present invention relates to a method for producing biologically active polypeptides from exogenous messenger ribonucleic acids (mRNAs) in a cytoplasmic extract from eukaryotic cells that have grown as monolayers. More precisely, there is provided a method for producing biologically active polypeptides from exogenous messenger ribonucleic acids (mRNAs) in a cytoplasmic extract efficient in translating mRNAs, which comprises the steps of: (a) growing eukaryotic cells in monolayer; (b) adding extraction buffer to the monolayer of step (a) and collecting the cells; (c) disrupting the cell membrane to obtain the cellular extract; (d) hydrolyzing endogenous mRNAs of the extract of step (c) using micrococcal nuclease and inhibiting the nuclease with EGTA or pTp to obtain a translational extract; (e) adding exogenous mRNAs and exogenous essential amino acids to the translational extract of step (d); and (f) incubating the translational extract of step (e) for a time sufficient for the translation of the exogenous mRNAs into the biologically active polypeptides while adding an energy regenerating system.

Description

PRODUCTION OF BIOLOGICALLY ACTIVE POLYPEPTIDES

BACKGROUND OF THE INVENTION

( a) Field of the Invention The invention relates to a method for producing biologically active polypeptides from exogenous messenger ribonucleic acids (mRNAs) in a cytoplasmic extract from eukaryotic cells that have grown as monolayers . (b) Description of Prior Art

In higher organisms, several developmental systems have been described where gene expression is thought to be controlled at the level of protein synthesis (translation) { Translational control . Edited by J. .B. Hershey, M.B. Matthews and N. Sonenberg, 1996, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.-Y, USA. 794 p.). In order to study the gene expression at this latter level, in vitro translation systems have been successfully generated. Due to its high translational activity with endogenous and exogenous messenger RNAs (mRNAs), its ease of preparation and the stability of the system on storage, the rabbit reticulocyte lysate system in its native form or after micrococcal nuclease treatment is probably the most widely used eukaryotic cell-free protein synthesizing system (Fresno, M. et al., 1976, Eur. J. Biochem. 68:355-364; Pelham, H.R.B. et al . , 1976, Eur. J. Biochem. 67:247-256). However, reticulocytes are not representative of eukaryotic cells in the way they regulate translation. They cannot or do not respond to various physiological (eg: hormones, toxines, ions, etc), chemical, and other external (heat shock, magnetic fields, etc) stimuli which are important regulators of cellular functions in nucleated cells, and they cannot be used for studies on viral infection. In addition to the widely used reticulocyte lysate and wheat germ systems (Clemens, M. J. 1979, In Transcription and translation : a practical approach (B. D. Hames & S. J. Higgins) . IRL Press pp. 231-270), extracts from many eukaryotic cell types have been prepared for the translation of exogenous mRNAs. Some of these systems are 10,000 to 30,000 x g supernatants which have been preincubated or nuclease- treated to eliminate endogenous mRNAs and then dialysed with SEPHADEX™ G-25 to standardize ionic conditions (Clemens, M. J. 1979, Tn Transcription and translation : a practical approach (B. D. Hames & S. J. Higgins) . IRL Press pp. 231-270; Mathews, M. B., 1972, Bioch . Biophys Acta 272:108-118). Other systems refer to a S-10 (1500 x g) cell-free extract that was initially obtained from mouse L cells for the translation of exogenous mRNAs (Skup, D. et al., 1977, Nucleic Acids Res . 1000:3581- 3587). When compared to the rabbit reticulocyte lysate, the major disadvantages of these systems are: i) their relative poor reinitiation in translational activity, ii) the large-scale (in liters) suspension cultures requirements that are needed for the production of cytoplasmic extracts efficient in protein synthesis, iii) the preparation procedures are specialized in the sense that they seem applicable only to the cell line for which the method was developed, iv) some of these extracts must be prepared freshly since there is a great loss of activity particularly of initiation on freezing.

Most of the synthesizing systems known to date rely on the production of polypeptides in bacteria, which as prokaryotic cells do not fulfill the translational requirements of higher eukaryotic cells (i.e. glycosylation) . SUMMARY OF THE INVENTION

In this patent proposal, a method for the production of polypeptides from cell-free extracts from eukaryotic cells that have grown in monolayers will be described. Some of the major advantages of this method are the following:

A) The cells can be grown by employing small volumes of cell culture medium before the generation of the translational extract.

B) 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.

C) The extract can be prepared by employing unexpensive compounds that are available from commercial sources. D) The cytoplasmic extract that is generated translates endogenous as well as exogenous added mRNAs with very high efficiencies, as seen with the incorporation of [ S Imethionine (or any other radiolabeled amino acid(s)) into newly synthesized polypeptides. Moreover, after hydrolysis of the endogenous mRNAs with micrococcal nuclease, an important synthetic activity on added exogenous mRNAs can be obtained with this in vitro system.

E) The proteins synthesized in vi tro in the cell-free extract that is not treated with micrococcal nuclease are an accurate reflection both qualitatively and quantitatively of the proteins synthesized by the whole cell prior to extract preparation.

F) Finally, the extract can be freezed and subsequently thawed for further use. In accordance with the present invention, there is provided a method for producing biologically active polypeptides from exogenous messenger ribonucleic acids

(mRNAs) in a cytoplasmic extract efficient in translating mRNAs, which comprises the steps of: a) growing eukaryotic cells in monolayer; b) adding extraction buffer to the monolayer of step a) and collecting the cells; c) disrupting the cell membrane to obtain the cellular extract; d) hydrolyzing endogenous mRNAs of the extract of step c) using micrococcal nuclease and inhibiting the nuclease with EGTA or pTp to obtain a translational extract; e) adding exogenous mRNAs and exogenous essential amino acids to the translational extract of step d) ; and f) incubating the translational extract of step e) for a time sufficient for the translation of the exogenous mRNAs into the biologically active polypeptides while adding an energy regenerating system.

In accordance with another embodiment of the present invention, there is provided a method which further comprises a step when the cellular extract of step f) may be diluted, adding a mixture of salts of the extract to maintain an efficient molarity for translation.

In accordance with another embodiment of the present invention, there is provided a method which further comprises a step wherein the cellular extract of step c) is centrifuged to collect the supernatant cytoplasmic extract. BRIEF DESCRIPTION OF THE DRAWINGS

Figs. 1, 2 and 4 illustrate each a fluorography of radiolabelled polypeptides synthesized in in vi tro protein synthesis in accordance with the method of the present invention using BHK cells;

Fig. 3 illustrates a fluorography of radiolabelled polypeptides synthesized in in vi tro protein synthesis in accordance with the method of the present invention using Rat 6 cells; and Fig. 5 illustrates the different enzyme addition protocol for maximum energy generation in accordance with the present method.

DETAILED DESCRIPTION OF THE INVENTION

MATERIALS AND METHODS

Cells

Eukaryotic cells are grown as monolayers in

Petri dishes (diameter, 100 mm) in their optimal cell culture medium. For example, baby hamster kidney (BHK) cells can be grown in Dulbecco ' s Modified Eagle Medium

(DMEM) supplemented with glutamine and 10% inactivated

(30 min., 56°C) fetal calf serum (FCS) or fetal bovine serum (FBS), or newborn calf serum (NBCS).

Preparation of the translational extract from eukaryotic cells. Micrococcal nuclease treatment

The method of preparation of this extract was adapted from a procedure described for the preparation of extracts from cells infected with rabbit poxvirus which catalyze in vi tro protein synthesis (Brown, G. D. et al., 1983, J. Biol . Chem. 258:14309-14314).

No starvation of an amino acid is required.

However, it might be necessary to radioactively label the newly synthesized polypeptides, prior to the preparation of the extract. For this, cell monolayers ^•7

(about 10 cells in the case of BHK cells; 100 mm dish) can be depleted of an amino acid, for example methionine, by incubating the cells in methionine-free cell culture medium for 30 min. at 37°C. The cells are then kept on ice for an additional 10 min. All subsequent steps are performed at 4°C (referred as "cold room" temperature) . The cells are washed with 30 mM Hepes-KOH [pH 7.4], 150 mM sucrose, 33 mM NH4CI, 7mM KC1, and are then permeabilized (for BHK cells: time of permeabilization is 90 sec; for NIH-3T3, a 60 sec. permeabilization is performed) with 100 μg/ l 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. After permeabilization, the solution is aspirated and the dishes are drained upright in order to aspirate the residual liquid. The cells are then scraped into 200 μl (Caeiro, F. et al., 1989, Virology 173:728-732) extraction buffer containing 100 mM Hepes-KOH [pH 7.4], 120 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM dithiotreitol, 2.5 mM ATP, 1 mM GTP, 1 mM spermidine, 20 mM creatine phosphate, 40 units of creatine phosphokinase per ml, and 40 μM of each essential amino acid except methionine. The cells are disrupted by passing them through a 25-gauge needle, or aby other convenient support or system, the minimal number of times needed to separate the nuclei. he translation extract, reffered 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 centrifugation are indicative and can be performed by varrying the parameters . To hydrolyse the endogenous mRNAs, 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- The reaction is stopped with the addition of 2.5 mM ethylene glycol-bis(β-aminoethyl ether )- N,N,N' ,N'-tetraacetic acid [pH 7] (EGTA) or of 2*- deoxythymidine 3 ' , 5 ' -diphosphate (pTp). 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 (ie: more or less concentrated) .

In vitro translation - Analysis of the translated polypeptides

Translation reaction (in 40 μl) contained 30 μl of cytoplasmic extract and 0.5 μCi/μl of

[³⁵S]methionine O1200 Ci/mmol) in order to radioactively label the newly synthesized polypeptides when convenient; the addition of exogenous mRNA and/or chemical compounds is performed accordingly when needed. RNAs can be obtained 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 structure (Bannerjee, A. K., 1980, Microbiol . Rev. 44:175-205).

In order to sustain the synthesis of polypeptides, the translational extract is supplied with exogenous amino acids (i.e. the 20 essential amino acids) after convenient time of translational incubation of the extract that is supplemented with the exogenous mRNA of interest to be translated. The extract is also supplied with the energy regenerating system, such as creatine phosphate, creatine phosphokinase, or both, after convenient time of translational incubation. If the exogenous added amino acids and energy regenerating system tend to dilute the translational extract, these compounds can be added with a mixture of the salts employed in the cytoplasmic extract in order to maintain a convenient molarity of the translational extract.

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 as described (Laemmli, U., 1970, Nature 227:680-685). Aliquots corresponding to equivalent amounts of protein are loaded.

RESULTS A method has been previously described for the generation of cytoplasmic extracts that were active for in vi tro transcription (Caeiro, F. et al . , 1989, Virology 173:728-732) and for in vitro translation (Brown, G. D. et al . , 1983, J. Biol . Chem. 258:14309- 14314). In both cases, the cells were grown in monolayers, permeabilized by using lysolecithin (L-α- lysophosphatidylcholine, palmitoyl; Sigma (Miller, M. R. et al., 1978, Biochemistry 17:1073-1080)), and the nuclei were removed by centrifugation after mechanical disruption of the plasma membrane. However, this method could only translate exogenous RNAs from viral source.

I have modified this protocol for the generation of a translational extract that was efficient in reinitiation of protein synthesis on endogenous as well as exogenous mRNAs of various sources. For this purpose, the cells were grown in monolayers, and disruption of the plasma membrane with lysolecithin was optimized for each cell type as described Caeiro, F. et al., 1989, Virology 173:728-7325). For example, incubation of the cells at 4°C for 90 seconds in washing buffer containing 100 μg of lysolecithine per ml is performed when BHK cells are employed. Moreover, the presence of 90 to 120 mM of potassium acetate in the translation reaction is generally found to be optimal for efficient incorporation of r[ 35S TJmethionine into newly synthesized polypeptides when the translational extract was obtained from this cell type.

Translation extracts were first employed without micrococcal nuclease treatment. The results reveal that the endogenous mRNAs are translated very efficiently. I have then assessed whether the extract could translate exogenous mRNA in presence of endogenous mRNA. Translations were programmed with various mRNAs such as: i) capped, polycistronic CAT(chloramphenycol acetyl transferase) -EMC( encephalomyocarditis ) -LUC

(luciferase) mRNA, which is containing both a 5' cap and an internal ribosomal entry site (such as described in

Pause, A.G.J. et al . , 1994, Nature 371: 762-767); ii) non-capped LUC mRNA from commercial source (Promega) ; iii) encephalomyocarditis (EMC) viral mRNA; iv) poliovirus mRNA with or without pretreatment of the extract with micrococcal nuclease. The results show that the exogenous mRNAs are efficiently translated.

It is of crucial importance to note that several single or multiple modifications can be introduced during the generation of the translational extract.

For example, the addition of hemine (up to 40 μM), the utilization of other concentrations of GTP (for example ranging between 0.05 and 2 mM) or creatine phosphate (up to 40 μM), the presence of additional tRNAs (for example, from calf liver and at 200 μg/ml), or higher concentrations of polyamines such as spermidine or spermine (for example, 10 mM), the pretreatment of the cells in hypertonic buffer (200 mM KC1 for 10 min at 37°C in FCS-supplemented DMEM, as proposed in reference 27), and the inhibition of micrococcal nuclease with 2 ' -deoxythimidine, 3'-5'- diphosphate (pTp; 0.12 mM) (Sigma) instead of EGTA also produce a translational extract that is efficient in reinitiation of protein synthesis.

DISCUSSION In accordance with the present invention, I have developed a cell-free translation system obtained from cytoplasmic extracts of cells permeabilized with lysolecithin for the study of translation of both endogenous as well as exogenous mRNAs and ultimately, for the production of biologically active polypeptides. My aim was to obtain a convenient protocol which is not time- and/or material-consuming. The original method for generation of such a translational extract, which was efficient for the reinitiation of translation on added exogenous RNAs, was described by Brown et al. (Brown, G. D. et al., 1983, J. Biol . Chem. 258:14309- 14314). These authors claimed that their system would be efficient for the reinitiation of protein synthesis on exogenous mRNAs; however, the RNAs that they employed were only from viral source. By following their initial protocol, I have never obtained the translation of added exogenous eukaryotic mRNAs obtained from other sources. Thus, changes were performed to obtain efficient reinitiation on these mRNAs. To this aim, the protocol described by Caeiro and Costa (Caeiro, F. et al . , 1989, Virology 173:728- 732), which was originally employed for in vitro transcription, was efficiently modified to optimize my in vitro translation system described above. 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). However, in certain circumstances, NH4CI or KC1 might be employed instead of potassium acetate, if desired. The optimal concentration of Mg 7+ 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. J. 1979, In Transcription and translation : a practical approach (B. D. Hames & S. J. Higgins) . IRL Press pp. 231-270). Thus, the optima should be established for every cell type and with each mRNA which is to be used. For example, I have found that the optimal concentrations in the extract obtained from BHK cells for translation of exogenous mRNAs was 1 mM of magnesium acetate and ranged between 90 to 120 mM of potassium acetate. Furthermore, I suggest that the translation reactions buffered with Hepes is preferable to Tris-HCl because of its lower pK and lack of sensitivity to temperature changes between 4°C and 30°C. However, any other efficient buffering system might therefore be employed to keep an optimal physiological pH.

It has been suggested that 200 μl of extraction buffer should be applied to cells growing on a 10 cm diameter petri dish to obtain the above-mentioned cytoplasmic extract. However, other sizes of petri dishes or supports such as spinner flasks can be employed; 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. For example, 800 μl of extraction buffer should be employed with a 20 cm diameter petri dish.

It has been mentioned above that the cytoplasmic extract (30 μl ) represented 75% of the final volume of the translational reaction (40 μl). These numbers are indicative and can be modified, if convenient, as follows : i) The final volume of the translational reaction can be changed by any factor of proportionality. For example, the reaction can be performed in 100 μl by employing 75 μl of cytoplasmic extract. ii) 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. For example, the translation reaction can be performed in a final volume of 40 μl by employing 15 μl of cytoplasmic extract; in this case, the concentration of the components that are present in the extraction buffer described above must be doubled.

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. (i) Hemine regulates the heme-regulated eIF-2 kinase (HRI) (Chen, J. -J. et al., 1991, Proc . Natl . Acad. Sci . USA 88:315-319; Chen, J. -J. et al . , 1991, Proc . Natl . Acad. Sci . USA 88:7729-7733) in rabbit reticulocyte lysates by promoting intersubunit disulfide bond formation in HRI, and by inhibiting the autokinase and eIF-2α kinase activities of HRI (Chen, J. -J. et al., 1989, J. Biol . Chem. 264:9559-9564; Yang, J. M. et al., 1992, J. Biol . Chem. 267:20519- 20524). Phosphorylation of the eukaryotic initiation factor 2 on its α subunit results in the binding and sequestration of guanine nucleotide exchange factor (eIF-2B) and leads to the cessation of the reinitiation of protein synthesis (Merrick, W. C, 1992, Microbiol . Rev. 56:291-315; Pain, V. M., 1986, Biochem. J. 235:625-637). It has been mentioned that hemine could also maintain reinitiation of protein synthesis in non- erythroid cell extracts (Ochoa, S. et al., 1979, Ann. Rev. Biochem. 48:549-580). Thus, hemine could be employed during preparation or incubation of the translational extract.

(ii) 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, Tn 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 [as described in Bader, M. et al . , 1986, Eur. J. Biochem. 75:103-109; Weber, L. A. et al . , 1975, Biochemistry 14:5315-5321, respectively] .

(iii) The energy regenerating system used to regenerate ATP relies on the presence of creatine phosphate and creatine phosphokinase (Ochoa, S. et al . , 1979, Ann. Rev. Biochem. 48:549-580). Thus, to delay a (possible) depletion in endogenous ATP pools, translations are suggested to be performed in presence of 40 μM of creatine phosphate as described (Morley, S. J. et al., 1985, Bioch . Biophys . Acta 825:45-56). iv) There are alternative energy-regenerating systems which can be employed in eukaryotic cell-free systems. The following energy regenerating systems might also be employed successfully: a) the pyruvate kinase and phosphoenol pyruvate system. This has the advantage of converting ADP to ATP and GDP to GTP directly

(whereas creatine phosphokinase and creatine phosphate rely on the presence of nucleoside diphosphate kinase and ATP endogenous to the cell extract) . b) It may also be possible to utilise endogenous enzymes of glycolysis to generate ATP if fructose 1, 6-biphosphate is added, provided this pathway is sufficiently active in vi tro to maintain the ATP level, (i) Micrococcal nuclease could have a deleterious effect on endogenous tRNAs (Pelham, H.R.B. et al., 1976, Eur. J. Biochem. 67:247-256). Thus, the addition of calf liver tRNA up to 200 μg 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).

(vi) The presence of polyamines could stimulate amino acid incorporation (Atkins, J. F. et al . , 1975, J. Biol . Chem. 250:5688-5695); thus, translation reactions can also be performed in the presence of spermine or spermidine (2 mM for example).

(vii) Preincubation of the cells in hypertonic medium induces resistance to a number of agents that interfere with polypeptide chain initiation (Bader, M. et al., 1986, Eur. J. Biochem. 75:103-109; Yates, J. R. et al., 1982, J. Biol . Chem. 257:15030-15034); thus, the pretreatment of the cells in hypertonic medium prior the preparation of the translational extract can be performed; for example, by preincubating the cells for 10 min at 37°C in cell culture medium containing 200 mM KC1.

(viii) 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 . 1000:3581-3587). Thus, 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).

(ix) Other detergents can be employed to permeabilize the plasma membranes during the generation of the translational extract, as described elsewhere (Neugebauer, J.M., 1990, Mehods Enzymol . 182:239-253); for example, digitonin or TRITON™ X-100 could be employed instead of lysolecithin.

(x) The cells may be depleted of (an) amino acid(s) other than methionine (as descibed above) by preincubating it with medium lacking (an)other amino acid(s). For example, 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

(xi) 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) to the cytoplasmic extract prior to its freezing. For example, use of up to 4% (vol/vol) of DMSO in the translational extract has no deleterious effect on translational efficiency after thawing of the extract.

(xii) The translational extract could also be dialysed or passed over SEPHADEX™ (coarse) at 4°C to lower the concentrations of amino acids and to standardize the ionic conditions before storage.

(xiii) To increase the translatability of exogenous added RNAs in the translational extract, it might be useful, in certain circumstances, to preincubate the cells in fresh cell culture medium (for example, by changing the medium 2 hours prior to the preparation of the translational extract).

(xiv) The optimal temperature for in vitro translation is 30°C (Clemens, M. J. 1979, In Transcription and translation : a practical approach (B. D. Hames & S. J. Higgins). IRL Press pp. 231-270). However, any other temperature giving an efficient translation might also be employed (for example, 37°C, as suggested in Clemens, M. J. 1979, In Transcription and translation: a practical approach (B. D. Hames & S. J. Higgins). IRL Press pp. 231-270).

(xv) Amino acids concentrations of about 40 μM each (less for the radiolabelled amino acid) are generally adequate in extracts from cultured cells (Clemens, M. J. 1979, In Transcription and translation : a practical approach (B. D. Hames & S. J. Higgins). IRL Press pp. 231-270). However, if there is little danger to run out of an amino acid during cell-free protein synthesis, these concentrations might be increased in consequence. Moreover, these concentrations can be reduced if the same results are obtained when compared to the use of 40 μM of each amino acid.

(xvi) It might be technically possible to couple in vi tro transcription/translation in the system described in this patent proposal. For this, transcription is dependent of exogenous added deoxyribonucleic acid (DNA) templates. This relies on the use of a transcription system, such the T3 , or T7, or SP6 RNA polymerase systems for example. 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.

The present invention will be more readily understood by referring to the following examples which are given to illustrate the invention rather than to limit its scope.

EXAMPLE I

Autoradiographies

Fluorography illustrated in Fig. 1

Baby hamster kidney (BHK) cells that have grown as monolayers were employed for in vi tro 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 (-).

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 was treated with micrococcal nuclease and employed for translation without exogenous added RNA: the endogenous RNA has been efficiently hydrolysed, since no polypeptides are visible (column 2). 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). In this typical experiment: 0.2μg of CAT-EMC-LUC RNA was employed; and 0.4 μg 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.

Other columns shown on this fluorography must not be taken into consideration.

Fluorography illustrated in Fig. 2

A reaction was performed with BHK cytoplasmic extract that was not treated with micrococcal nuclease either a) in absence of exogenous added RNA (column 2), or b) in presence of non-capped LUC RNA (0.2 μg) from commecial source (Promega) (column 3). Column 3 reveals that the LUC polypeptide is efficiently translated from non-capped RNA. When the extract is treated with micrococcal nuclease, the exogenous added LUC RNA is also efficiently translated (column 4). Positions of actin and tubulin are indicated.

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).

Fluorography illustrated in Fig. 3 Rat 6 (R6) cells that have grown as monolayers were employed for in vi tro 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 (-).

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 employed without exogenous added RNA shows no radiolabeled polypeptide (column 4). When CAT-EMC-LUC RNA (0.2 μg ) is present in the translation reaction, the CAT polypeptide is translated (columns 2 and 5; with extract not treated or treated with micrococcal nuclease, respectively), but the LUC polypeptide is below the limit of detection in this particular experiment (in the latter case, it seems that when confluent or overconfluent cells are employed for the generation of the cytoplasmic extract, sometimes it happens that the levels of cap-independent translation are reduced) .

BMV RNA is efficiently translated (columns 3 and 6; with extract not treated or treated with micrococcal nuclease, respectively).

Fluorography illustrated in Fig. 4

Baby hamster kidney (BHK) cells that have grown as monolayers were employed for in vitro 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 (-).

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). When CAT-EMC-LUC RNA (0.2μg) is present in the translation reaction, both the 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: encephalomyocarditis virus (EMC) RNA (0.2 μg; columns 2 and 5; with extract not treated or treated with micrococcal nuclease, respectively); poliovirus RNA (0.4 μg; columns 7; with extract that has been treated with micrococcal nuclease).

Discussion

Taken together, the results shown in fluorographies, illustrated in Figs. 1 to 4, present evidence that the experimental procedure that was employed for the generation of a cytoplasmic extract from cells that have grown as monolayers allows efficient translation of exogenous added RNAs such as: 1) capped RNA, 2) non-capped RNA,

3 ) RNAs containing an internal ribosome entry site MIRES),

4) brome mozaic virus (BMV) RNA.

It is yet not known, whether this system would allow efficient ribosomal frameshifting events (29, pp.522-523 and 653-684). EXAMPLE II

Different enzyme addition protocol for maximum energy generation in accordance with the present method 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 μl of creatine kinase (at 48 mg/ml that is resuspended in 50% (vol. /vol.) glycerol) is added to 200 μl of cytoplasmic extract.

However, it is also possible, if convenient, to change these conditions by: a) adding different volumes and/or concentrations of creatine kinase in the cytoplasmic extract (for example, 2 μl of 20 mg/ml (in 50% (vol. /vol.) glycerol) to 200 μl of cytoplasmic extract; b) to add the creatine kinase in the extraction buffer prior to the generation of the cytoplasmic extract; c) as described above, to add creatine kinase once the cytoplasmic extract has been generated; d) to add convenient volume and concentration of creatine kinase in each translation reaction to be performed, inserted of adding the enzyme to the extraction buffer or the cytoplasmic extract as described above; e) to employ pyruvate kinase (EC 2.7.1.40) instead of creatine kinase; f) to employ any other enzyme for the energy regenerating system which would allow efficient in vi tro protein synthesis; and g) to employ the full-length or the biologically active portion of a kinase that would act efficiently in the energy regenerating system. It is crucial that the enzymes that compose the energy regenerating system must be added freshly from a stock that preserves the biological activity of the enzyme. When a cytoplasmic extract is frozen in presence of creatine kinase (for example), and defrozen for further use, the cytoplasmic extract is efficient in translating endogenous RNAs, but fails to initiate translation or exogenous added RNAs. As said above, the enzyme must be added freshly to the cytoplasmic extract.

Thus, it is possible to prepare cytoplasmic extracts and to freeze them (for example, at -20°C, or on dry ice) for further use; once defrozen, the cytoplasmic extract must be supplied with creatine kinase or pyruvate kinase from a biologically active frozen stock (usually, these enzymes are stored at -20 °C in convenient concentrations of glycerol) to generate an efficient energy regenerating system.

Claims

I CLAIM:

1. A method for producing biologically active polypeptides from exogenous messenger ribonucleic acids (mRNAs) in a cytoplasmic extract efficient in translating mRNAs, which comprises the steps of: a) growing eukaryotic cells in monolayer; b) adding extraction buffer to the monolayer of step a) and collecting the cells; c) disrupting the cell membrane to obtain the cellular extract; d) hydrolyzing endogenous mRNAs of the extract of step c) using micrococcal nuclease and inhibiting the nuclease with EGTA or pTp to obtain a translational extract; e) adding exogenous mRNAs and exogenous essential amino acids to the translational extract of step d) ; and f) incubating the translational extract of step e) for a time sufficient for the translation of the exogenous mRNAs into the biologically active polypeptides while adding an energy regenerating system.

2. The method of claim 1, which further comprises a step when the cellular extract of step f) may be diluted, adding a mixture of salts of the extract to maintain an efficient molarity for translation.

3. The method of claim 1, which further comprises a step wherein the cellular extract of step c) is centrifuged to collect the supernatant cytoplasmic extract.