DE19503685A1 - Storage-stable reaction mixt. contg. many enzymes, reactants and stabilisers - Google Patents

Abstract

Prodn. of complex, multi-enzyme, storage-stable reaction mixt. (A) comprises combining in aq. soln.: (1) a natural or artificial, enzymatically active protein mixt., (2) reaction components and (3) a stabiliser (I) that: (a) does not affect, or increases, reactive capacity of the multi-enzyme system and (b) protects unstable reaction components against loss of biological activity during the process of stabilisation and storage. The mixt. is opt. frozen, vacuum dried and opt. overlayered with inert gas or mineral oil for storage at 0-10 deg C (not overlaid) or 20-30 deg C (if overlaid). The amt. of (I) required to increases activity of the mixt. is equiv. to that required to stabilise it.

Description

The invention relates to a process for the production of complex multi-enzymatic storage-stable reaction mixtures of native and optionally artificial enzymatic active protein mixtures, which are completed ready to use and without loss of activity 4 ° -10 ° C or if necessary at room temperature, as well as the use of these reaction mixtures for the synthesis, modification or analysis of polypeptides and Nucleic acids.

The use of cell extracts and enzyme mixtures to study complex biochemical reaction processes play a central role in molecular biology, biochemistry and molecular diagnostics.

Cell-free protein biosynthesis using lysates from wheat germ, Rabbit reticulocytes or Escherichia coli cells is a key method in that Research into basic mechanisms of synthesis, folding and post-translational Maturation of proteins and polypeptides. Problems of intracellular targeting and maturation of proteins and other biomolecules with the help of microsomes (ER-containing Cell fraction), isolated mitochondria, chloroplasts or nuclear extracts.

In addition to applications from basic research, multi-enzymatic cell extracts for protein biosynthesis are becoming increasingly important for the solution of preparative-synthetic or diagnostic objectives ( 21 ; 22 ). In-vitro translation on a semi-preparative scale is a promising tool for the synthesis of protein fragments for mapping epitopes, catalytic centers or artificial antigen determinants. There is also a trend towards the use of multi-enzymatic reaction systems in molecular biological methods such as RNA synthesis in vitro ( 7 ), polymerase chain reaction (PCR) ( 2 ; 5 ) or DNA sequencing ( 20 ). The performance and synthesis accuracy of DNA & RNA polymerases can be increased by the combination with other enzymes and cofactors such as inorganic pyrophosphatase, DNA binding proteins, RNase inhibitors or exonucleases. Demanding applications such as the amplification of long DNA fragments (<10 kb) with high synthesis accuracy (LA-PCR), starting from genomic DNA, are only possible through the combination of several DNA polymerases with different properties ( 2 ; 5 ).

The wide application of complex multi-enzymatic reaction systems is still limited in applied research and diagnostics, because according to the current state of the art problems of stability, simple handling and thus reproducibility are only inadequately solved. The decisive disadvantage of complex multi-enzymatic reaction systems compared to reaction mixtures with single enzymes is their complicated technical handling. The complete reaction mixtures with all enzymes, substrates and cofactors are stable as an aqueous solution neither at room temperature nor at -20 ° C. A problem that affects the stability of multiencymatic reaction mixtures arises from the fact that the optimal reaction conditions (pH, ionic strength, type of salts, concentration of the stabilizing agent) for the biosynthesis mostly deviate from those which are necessary for the permanent preservation of the Enzyme components and cofactors are optimal ( 10 ; 12 ; 18 ). In addition, the individual protein components of cell extracts or enzyme mixtures, depending on whether they are soluble enzymes, fibrillary structural proteins, membrane-associated enzyme complexes or nucleoproteins, place different demands on the storage conditions in solution. Traditionally, commercial reaction systems for in-vitro transcription, translation, PCR or DNA sequencing are therefore offered in the form of kits, in which the components are provided individually. In the worst case, as the following example shows with a reaction mixture for in vitro translation, the various components of a kit must be stored separately under different conditions after receipt. The task of the user is to mix the reaction mixture from a large number of individual components for each test sample. This process is difficult to automate. The number of samples increases the amount of time and the likelihood of errors, and this affects the reproducibility. The preparation of the reaction mixtures often takes more time than the actual test execution.

This problem can be exemplified by the composition of the reaction mixtures for represent in-vitro translation using cell-free extracts:  

Due to the different temperature requirements of -85 ° C, -20 ° C and 4 ° C, the technical effort for the storage and transport of the starting components of the Translation mixture high. Components 1 and 4 are only a few even at a constant -20 ° C Stable for months and then have to be replaced by fresh solutions. The dispatch of Cell extracts and the other reaction components in soluble form must be in dry ice to avoid thawing the sample. In this respect, storage is not unproblematic than having expensive freezers, usually with liquid nitrogen operated, with technically complex safeguards and alarm systems required.

The most sensitive and most important part of reaction mixtures for in-vitro translation are cell extracts because they contain all the enzymatic components for ribosomal protein biosynthesis. The complex biochemical reaction sequence of the mRNA-controlled polypeptide synthesis requires the cooperative interaction of a large number of enzymes, enzyme complexes and structural proteins with different structures and stability. Unlike in cells, where the proteins of the ribsomal translation apparatus form macromolecular complexes, which are stabilized by interactions with the filamentous components of the cytoskeleton, the cell-free extracts active in translation no longer contain an intact cytoskeleton. Free in solution, the macromolecular enzyme complexes can dissociate very easily and thus lose their reactivity. The only reliable method for the preservation of soluble cell extracts according to the current state of the art is storage in the deep-frozen state at -80 ° -85 ° C. Nevertheless, there are a number of problems with it. When frozen, the cell extracts from wheat germ, reticulocytes, or E. coli cells lose part of their enzymatic activity, since many proteins are irreversibly damaged by the formation of water crystals ( 12 ). Repeated freezing and thawing of the cell-free extracts for successive applications leads to complete loss of their activity in preparations from E. coli and rabbit reticulocytes, while according to our own studies the translational activity of wheat germ extracts is reduced by 30-50% after each freezing and thawing.

The instability of cell-free extracts against repeated freezing and thawing forces the user to an uneconomical consumption of this expensive reaction component. For reproducible test results are only allowed for each batch of the translationally active cell extract be thawed once. The activity losses during storage in the frozen state are higher if, instead of the highly concentrated cell extract, the complete, ready-to-use Translation mixture freezes. After just one freeze and one week of storage A complete reaction mixture loses about 60% of the enzymatic activity, measured on the final yield of the translation product compared to a reaction mixture that is fresh was put together from the individual components. Apparently the protein concentration plays which is 3-4 times higher in the undiluted cell extract than in the finished translation mixture, the crucial role for the stability of the multi-enzymatic components during the Freezing and frozen.

The stability problem with other reaction mixtures for the synthesis and modification of nucleic acids behaves similarly. Even freezing once in aqueous solution completely deactivates RNA and DNA polymerases, so that they can only be stored at -20 ° C in stable form as single enzyme solutions with the aid of cryoprotectors. Glycerin is usually used in a concentration of at least 50% as an auxiliary, which prevents the formation of harmful water crystals. Due to the necessary high concentration of glycerin or other auxiliary substances such as dimethyl sulfoxide, polyethylene glycol or bovine serum albumin, cryoprotectors cannot be used for the deep-freeze storage of complete reaction mixtures ( 12 ; 6 ). The viscosity of the resulting solutions affects the reactivity of the enzymatic components and is a problem for subsequent analyzes of the reaction mixture. At concentrations <50%, the storage stability of the enzymes also drops at -20 ° C.

Surprisingly, little has changed in the technology for the production and preservation of enzymatically active cell extracts since the first publications on cell-free protein biosynthesis and post-translational modification in the mid-1970s. In principle, the alternative to stabilizing the storage by freeze-drying offers itself to the already described state of the art. This method is primarily used to stabilize liposomes and membrane fractions ( 6 ), single enzyme preparations ( 1 ; 4 ; 8 ) or reaction mixtures without enzyme components ( 16 ), using special sugars or other polyols in combination with bivalent metal ions or surfactants ( 13 ; 14 ; 15 ) prevent the denaturation of the enzymes or membrane structures due to water loss. The natural sugar trehalose has proven to be particularly effective for stabilizing biomaterials during freeze drying ( 19 ; 9 ; 10 ). This sugar is enriched by some organisms, the so-called cryptobionts, in extremely dry conditions in the cells, thus ensuring the survivability of these organisms in the event of total water loss ( 17 ; 3 ).

The technical problems underlying the present invention are the provision a manufacturing process that overcomes the disadvantages of the prior art in terms of stable Storage, transport and ready-to-use preparation of multi-enzymatic Reaction mixtures for the implementation of complex biochemical or molecular biological Avoids reactions. In particular, the method is intended to freeze the stored and can dispense with samples to be transported, and by providing complete, d. H. already provided with all necessary reaction components reaction mixtures, the Reduce experimental time expenditure for the user considerably and the reproducibility increase. The object is achieved by a method with the features of Claims 1 and 12. The subclaims relate to special embodiments of the inventive method.

The present invention differs from the prior art for the use of Trehalose as a stabilizer for the first time in that complete and multi-enzymatic Reaction mixtures with many protein components and not individual enzymes or Reaction part mixtures without enzymatic components are stable in storage and ready for use can be produced. Second, conditions were found under which the trehalose ensures sufficient storage stability without additional auxiliaries and additionally the Responsiveness of the multi-enzymatic reaction mixture increased after reconstitution.

The method according to the invention relates to multi-enzymatic reaction mixtures for cell-free protein biosynthesis (in vitro translation). Under various conditions, 50 ul translation mixtures were vacuum dried and reconstituted in water. The reactivity of the reaction mixtures treated in this way was demonstrated by the translation of synthetic obelin mRNA. Obelin is a luminescent protein with a molecular weight of 20 kDa. The translation product was detected both quantitatively by measuring the TCA-precipitable radioactively labeled proteins from 5 μl of the translation mixture, and qualitatively by electrophoretic analysis in SDS-PAG with subsequent radioautography, after 2 hours of incubation at 25 ° C. It was found that the protein components of the translationally active cell extract completely denature during the drying process, regardless of whether they were previously frozen or not. The proteins no longer dissolved from the lyophilized residue in water and remained in the reaction mixture as cloudy precipitate. A radiolabelled translation product (obelin) could not be detected ( Fig. 1 / A, lanes 4 and 8), although the TCA-precipitable radiolabelled proteins could be detected ( Fig. 1 / B). This effect was based on the non-specific formation of the [³⁵S] L-methionine used for the radioactive labeling on the denatured proteins.

Surprisingly, it was found that even low trehalose concentrations (2.5% and 5%) without additional additives in the translation mixture are sufficient to at least partially protect the sensitive enzymatic components of the cell-free wheat germ extract from denaturation during vacuum drying and reconstitution ( Fig. 1 / A & 1 / B). The best effect could be achieved with a final trehalose concentration of 10%. About 84% of the original translation activity, measured in terms of the synthesis yield, could be obtained after lyophilization and reconstitution in comparison with the control reaction with a freshly prepared reaction mixture without trehalose. Interestingly, it emerged from the first experiments that the stabilizing effect of trehalose does not correlate proportionally with the concentration in the translation mixture.

In 25 µl, 50 µl and 100 µl reaction mixtures with 10% trehalose, which were either lyophilized or simply vacuum dried, the kinetics of obelin translation after reconstitution in water were determined and compared with the synthetic kinetics in the control reaction. A freshly prepared translation mixture without trehalose with the same reaction volume was used as the control reaction. In order to determine the translation kinetics of obelin, 3 µl samples were taken from the translation mixture at certain intervals and the TCA-precipitable radioactively labeled protein fraction was quantified therein (in cpm). It was shown that in the 25 µl reaction batches, the kinetics of the synthesis of the obelins in the differently treated translation mixtures did not differ significantly from one another or from the kinetics of the control reaction ( Fig. 2). In other words, 25 µl translation mixtures can be made storage-stable by vacuum drying or reconstituted without losing their original reactivity, it being irrelevant whether they are snap-frozen in an alcohol / dry ice bath beforehand.

In contrast, with 50 µl and 100 µl translation mixtures the conditions of storage stabilization influenced the effectiveness of the preservation of the translation activity ( Fig. 3 / A & 3 / B).

The differences can be seen both in the course of the kinetics and in the difference in the synthesis yield of the radioactively labeled translation product after 2 hours of incubation. When the 50 .mu.l reaction mixtures were made stable by simple vacuum drying (2.5 hours), about 40% of the original translation activity was lost in comparison to the control reaction. Freezing the samples at -50 ° C before vacuum drying (lyophilization) drastically reduces the loss of activity of the 50 µl translation mixtures during the drying process. The translational activity of the reconstituted lyophilized reaction mixtures is almost 100% compared to the control reaction in a fresh translation mixture without trehalose. The situation is similar with the 100 µl translation mixtures ( Fig. 3 / B).

Surprisingly, the trehalose in the same concentration, which also ensures the best preservation of the reactivity during storage stabilization and reconstitution, stimulated the in vitro translation in untreated freshly prepared 50 µl and 100 µl reaction mixtures ( Fig. 3 / A & 3 / B). The synthesis yields were about 10-20% higher than in the corresponding control reactions without trehalose. A positive effect of trehalose on ribosomal protein biosynthesis was previously unknown. The increase in the translational activity of cell-free extracts by trehalose is particularly relevant for the method according to the invention, since this results in a loss of activity of 10-20% (compare the corresponding kinetics in Fig. 3 / A & 3 / B), which also occurs when the translation mixtures are lyophilized , is balanced again. For example, the reconstituted storage-stable reaction mixtures have the same translation activity as the translation mixtures without trehalose, which are composed of individual components according to the prior art.

A decisive advantage of the process according to the invention is the storage stability of the lyophilized, ready-to-use reaction mixtures. Several complete 100 μl translation mixtures were lyophilized with the addition of 10% trehalose, then sealed airtight and stored for 1 or 2 weeks at 4 ° -8 ° C. in a normal refrigerator or at room temperature (25 ° C.). The lyophilized reaction mixtures were then reconstituted in water, the kinetics of obelin translation were determined and compared with translation kinetics in a fresh reaction mixture ( Fig. 4). The results demonstrate that the translation activity of the reaction mixtures which have been made stable in storage is not impaired by storage at 4-8 ° C. Storage of the lyophilized translation mixtures at RT, however, led to a sharp drop in reactivity. The reason for this is that the hygroscopic acetate salts from the translation buffer are not included in the amorphous glass mass of the trehalose, but are deposited as a fluffy residue. This residue attracts the water from the air column above, so that the dried translation mixture is moistened. Too high a water content and a storage temperature of <10 ° C sets in motion enzymatic degradation processes that deactivate unstable substrates such as ATP and GTP and also sensitive enzymatic components. As a result, the dried translation mixtures lost up to 70% of their initial activity after just one week of storage at room temperature. This loss of activity could not be compensated for by adding fresh ATP and GTP when reconstituting the translation mixture. According to the invention, this problem can be solved by overlaying the lyophilized reaction mixtures with an inert gas.

The method according to the invention thus enables the ready-to-use preparation of native and artificial enzymatically active protein mixtures with reaction buffers, Cofactors and substrates in which:

• - The reaction mixtures in aqueous solution a stabilizing agent, which the one hand Responsiveness of the multi-enzymatic system increased or not impaired and on the other hand, the unstable reaction components before losing their biological Activity or its biological structure during storage stabilization storage protects,
• - if necessary after freezing
• - these are vacuum dried,
• - If necessary, an overlay with inert gas or mineral oil takes place.

The storage-stable reaction mixtures thus obtained are according to the invention Reconstitution in water by adding user-specific Key components depending on the desired enzymatic reaction for the synthesis, Modification or analysis of polypeptides or possibly nucleic acids used.

The complex multienzyme reaction mixtures produced according to the invention have the advantage that they at 0-10 ° C or, if necessary, after layering with an inert gas Ambient temperature (20-30 ° C) can be stored and transported stably. Thereby the technically and financially high expenditure for the acquisition and maintenance of one is eliminated Freezer equipment as required by conventional technology. your  second crucial advantage is that they are ready to use with all the necessary Reaction components are prepared so that the user only the desired reaction can start by adding one, maximum of two, key components. The enables the disadvantages of the prior art to be circumvented with respect to:

• - the simultaneous execution of a large number of parallel experiments,
• - high reproducibility of the enzymatic reactions,
• - a minimal amount of time to prepare for the experiment,
• - the avoidance of errors in the composition of reaction mixtures from many Components,
• - the automation of complex biochemical multi-enzymatic reaction processes.

The invention will be explained in more detail below using an example.  

Embodiment 1. Production of a translation-active reaction mixture on the basis a wheat germ lysate using the stabilizing agent Trehalose 1.1 Production of a ready-to-use, storage-stable translation mixture Individual components

On a ice bath at 0 ° -4 ° C, the ready-to-use translation mixture in a 1.5 ml Eppendorf reaction tube pipetted together from the following individual components:

• a) 4 ul master mix, consisting of 312 mM HEPES-KOH pH 7.6, 12.5 mM ATP, 1.25 mM GTP, 100mM creatine phosphate, 3.12mM and 25mM DTT;
• b) 2 µl amino acid mix, consisting of 2.5 mM of 19 natural L-amino acids without L-methionine (Alternatively, L-leucine or L-lysine may also be absent, depending on which of the Amino acids are used to label the translation product);
• c) 16 ul translation buffer consisting of 120 mM K acetate, 5 mM Mg acetate and 30% by weight trehalose;
• d) 3 µl RNase-free water (Millipore-18 MΩ);
• e) 2 µl creatine phosphokinase solution (1,125 mg / ml);
• f) 2 µl yeast tRNA (125 mg / ml);
• g) 2 µl RNSse inhibitor (40-200 U);
• h) 16 µl wheat germ extract, consisting of wheat germ lysate with a concentration of 90 A₂₈₀ / ml, 40 mM HEPES-KOH pH 7.6, 120 mM K-acetate, 5 mM Mg-acetate and 6 mM DTT.

The final volume of the finished translation mixture is 50 µl. For the production of 25 or 100 µl translation mixtures must be the volumes of the starting components be proportionally reduced or enlarged.

1.2 Storage stability of the translation mixtures

The components of the translation mixtures are carefully mixed in the reaction vessel and collected by pulse centrifugation at the bottom of the vessel. Immidiatly after are the reaction vessels with the translation mixtures in an open lid  Alcohol / dry ice bath (-50 ° C) snap frozen. After another minute, the Reaction tubes in a centrifuge with a connected vacuum pump and cold trap (SpeedVac or comparable devices) transferred and vacuum dried at a constant 30 ° C. Translation mixtures with a final volume of 30 µl are used without prior shock freezing vacuum dried. Centrifugation during vacuum drying ensures that the enzymatic and non-enzymatic components of the translation mixture on the ground of the reaction vessel and drop-shaped into the glassy mass of the trehalose be sintered.

After 2.5 to 3 hours, the drying process is ended by the Vacuum centrifuge carefully ventilated. The air inlet on the centrifuge is with a Sterile filter to provide the contamination of the dried reaction mixtures with to prevent microbial germs. The test tubes with the dried ones Translation mixtures are removed from the centrifuge under sterile conditions and immediately sealed airtight.

In this state, the dried translation mixtures at 0 ° C-10 ° C in Refrigerator be stored stably. For storage at temperatures between 20 ° -30 ° C the dried reaction mixtures with nitrogen or another inert gas, immediately after removing the still open reaction vessels from the vacuum centrifuge were overlaid.

1.3 Reconstitution of the vacuum-dried translation mixtures and implementation in vitro translation

Before carrying out the in vitro translation, the vacuum dried ones Reaction mixtures brought into solution with water. First, the vacuum dried Translation mixtures put on ice. Depending on the respective final reaction volume exactly defined volumes of water are pipetted onto the vacuum-dried residues. For a 50 µl translation batch, this corresponds to a volume of 32 µl, for 25 µl and 100 µl translation approaches corresponding to 16 µl and 64 µl. By carefully mixing the glassy trehalose drops with the reaction components completely dissolved. While From time to time, the translation mixtures should always be put on hold to dissolve to keep them cool. Overall, the transfer of the storage-stable Translation mixtures in solution no more than 5 minutes.  

The translation reaction is achieved by adding 4 µl (1 µg / µl) mRNA solution, or correspondingly 2 or 8 µl mRNA solution for the reaction volumes 25 and 100 µl, and 1 µl or 0.5 or 2 µl [³⁵S] L -Methionine 1000 Ci / mmol started. The stated amounts of the mRNA relate to the luminescent enzyme obelin translated in the case example. For other proteins, the optimal amount of mRNA must be determined empirically in titration test series in the range of 1-10 µg for a 50 µl translation batch, but the volume of the RNA solution added must remain constant. After the addition of the mRNA and the radioactively labeled amino acid, the translation mixtures are mixed briefly, collected by pulse centrifugation at the bottom of the vessel, and then incubated at 25 ° C. in a thermostat. After 0, 15, 30, 60, 90, 120 and 180 minutes, 2 µl samples are taken from the reaction mixtures, dropped onto filter disks made of Whatman 3 MM chromatography paper and air-dried. The radioactively labeled translation product obelin is detected in a conventional manner by precipitation of the samples on the filter paper and subsequent washing of the precipitated samples in ice-cooled 5% trichloroacetic acid and acetone. To determine the acid-precipitable radioactivity (in cpm) as a measure of the amount of the synthesized translation product, the filter disks with the treated samples are then immersed in 3 ml of a corresponding scintillation mixture and measured on a corresponding scintillation counter. Taking samples from the translation mixture at different times enables the kinetics of the translation process to be determined. Alternatively, the detection of the translation product can also be carried out qualitatively by separating the samples on a 12% SDS-PAG and then detecting the labeled translation product by direct radioautography with an X-ray film.

Translation mixtures without trehalose for the control reaction to demonstrate the influence of the stabilizing agent on the translational activity and storage stability of the Translation mixtures are set up according to the scheme above, but under Use of a translation buffer (component c) without trehalose.  

literature

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Claims (15)

1. A process for the preparation of complex multi-enzymatic storage-stable reaction mixtures, characterized in that native or artificial enzymatically active protein mixtures with reaction components and a stabilizing agent which on the one hand does not impair and possibly increase the reactivity of the multi-enzymatic system and which on the other hand increases the unstable reaction components before the loss of their biological activity or their biologically active structure during storage stabilization and storage, can be combined in aqueous solution, optionally frozen, by vacuum drying and then, if appropriate, covered with an inert gas or mineral oil in a at 0 ° -10 ° C or 20 ° -30 ° C storage-stable state are transferred, the amount of stabilizing agent for increasing the reactivity of the multienzymatic reaction mixture is equivalent to the amount for the camp stabilization of the complex multi-enzymatic reaction mixture is required. 2. The method according to claim 1, characterized in that one is native enzymatic uses active protein mixtures cell extracts or fractions from these. 3. The method according to claim 1, characterized in that one as an artificial enzymatic active protein mixtures a mixture of pre-purified single enzymes, cofactors and optionally uses structural proteins. 4. The method according to claim 1, characterized in that one as the reaction components enzymatic and non-enzymatic cofactors, enzyme substrates, nucleotides and Nucleosides or their oligomers, proteins, peptides, thiol compounds, RNA, DNA and where appropriate, derivatives of each of the above substances are used individually or in combination. 5. The method according to claim 1, characterized in that the stabilizing agent is chemically inert sugar or, if appropriate, a sugar mixture, which or dried state an amorphous glass mass with a residual water content of no more forms as 0.2 g H₂O / g dry matter. 6. The method according to claim 1 and 5, characterized in that the concentration of Stabilizing agent in the ready-to-use reaction mixture in aqueous solution is not less than 5% by weight and not more than 10% by weight. 7. The method according to claim 1, 5 and 6, characterized in that the Stabilizing agent is trehalose.   8. The method according to claim 1, characterized in that the vacuum drying in one commercial vacuum centrifuge (e.g. SpeedVac) at 30 ° C up to a Residual water content of 0.2 g H₂O / g, but at least 2.5 hours is carried out. 9. The method according to claim 1, characterized in that reaction mixtures with a Volume 30 µl before vacuum drying at -50 ° C in an alcohol / dry ice bath or if necessary, be frozen in liquid nitrogen at -80 ° C. 10. The method according to claim 1 and 8, characterized in that reaction mixtures with a volume <30 µl can be vacuum dried directly from the solution. 11. The method according to claim 1, characterized in that nitrogen as the inert gases, Helium or argon can be used. 12. Use of the complex multi-enzymatic storage-stable reaction mixtures after Claims 1 to 11 for the synthesis, modification or analysis of polypeptides or Nucleic acids after reconstitution in water, each one or more specific Key components are added. 13. Use according to claim 12, characterized in that the key components radioactive or non-radioactive labeled amino acids or their corresponding aminoacyl-transfer RNA molecules, radioactive or non-radioactive labeled nucleotides or, if appropriate, their derivatives or oligomers, natural or artificial messenger RNA, DNA of various origins or combinations of the above substances. 14. Use according to claim 12 and 13 for cell-free ribosomal protein biosynthesis and optionally for the post-translational modification of peptides, polypeptides and Proteins. 15. Use according to claim 12 and 13 for replication, reverse or non-reverse Transcription, possibly enrichment or modification of nucleic acids.