DE19503685C2 - Process for the preparation of complex multi-enzymatic storage-stable reaction mixtures and their use - Google Patents

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 win for protein biosynthesis is becoming increasingly important for the solution of preparative-synthetic or diagnostic objectives (21; 22). The in vitro translation is on a semi-preparative scale a promising tool for the synthesis of protein fragments for mapping Epitopes, catalytic centers or artificial antigen determinants. Even with the molecular biological methods, such as RNA synthesis in vitro (7), the Polymerase chain reaction (PCR) (2; 5) or DNA sequencing (20), is a trend towards Use of multi-enzymatic reaction systems. The performance and Synthesis accuracy of DNA & RNA polymerases can be achieved by combining them with others Enzymes and cofactors such as inorganic pyrophosphatase, DNA binding proteins, RNase inhibitors or exonucleases can be increased. Demanding applications like that Amplification of long DNA fragments (<10 kb) with high synthesis accuracy (LA-PCR), starting from genomic DNA, is only possible by combining several DNA Polymerases with different properties possible (2; 5).

The widespread use of complex multi-enzymatic reaction systems are in the applied research and diagnostics are still limited because after the current state of the art problems of stability, ease of use and thus reproducibility are insufficiently resolved. The crucial disadvantage of complex  multi-enzymatic reaction systems versus reaction mixtures with single enzymes consists in their complicated technical handling. The complete reaction mixtures with All enzymes, substrates and cofactors are as an aqueous solution neither at room temperature still stable at -20 ° C. A problem affecting the stability of multi-enzymatic Reaction mixtures impaired arises from the fact that the optimal Reaction conditions (pH value, ionic strength, type of salts, concentration of the Stabilizing agent) for the biosynthesis usually deviate from those for the permanent Preservation of the enzyme components and cofactors are optimal (10; 12; 18). Moreover represent the individual protein components of cell extracts or enzyme mixtures, depending on whether it is soluble enzymes, fibrillary structural proteins, membrane-associated enzyme complexes or nucleoproteids, different requirements for storage conditions in Solution. Traditionally, therefore, commercial reaction systems for in vitro Transcription, translation, PCR or DNA sequencing are offered in the form of kits in which the components are provided individually. In the worst case, like the following example with a reaction mixture for in vitro translation shows the different Components of a kit are stored separately under different conditions after receipt become. It is the responsibility of the user to determine the reaction approach for each test sample a large number of individual components. This process is difficult automatable. With the number of samples, the time required and the Probability of errors, and this affects reproducibility. Often the preparation of the reaction mixtures takes more time than the actual one Test execution.

This problem can be exemplified using the composition of the reaction mixtures for 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 the cell extracts because they are all the enzymatic components for the ribosomal Contain protein biosynthesis. The complex biochemical reaction sequence of the mRNA-controlled Polypeptide synthesis requires the cooperative interaction of a variety of enzymes Enzyme complexes and structural proteins with different structure and stability. Different to in cells where the proteins of the ribsomal translator are macromolecular complexes form, which is stabilized by interactions with the filamentous components of the cytoskeleton the cell-free active extracts no longer contain an intact cytoskeleton. Free in The macromolecular enzyme complexes can dissociate and lose solution very easily hence their responsiveness. The only reliable method for preserving Soluble cell extracts according to the current state of the art are stored in the frozen state at -80 ° -85 ° C. Nevertheless, there are a number of problems associated with this. When frozen, the cell extracts from wheat germ, reticulocytes, or E. coli cells lose part of their enzymatic activity because of the formation of water crystals many proteins  are irreversibly damaged (12). Multiple freezing and thawing of the cell-free extracts for successive applications leads to preparations from E. coli and Rabbit reticulocytes to completely lose their activity while undertaking their own Examines the translational activity of wheat germ extracts by 30-50% after each Freezing and thawing reduced.

The instability of cell-free extracts against repeated freezing and thawing forces the user to an uneconomical consumption of these expensive reaction components. 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 synthesis is similar and modification of nucleic acids. Even freeze once in aqueous solution completely deactivates RNA and DNA polymerases so that they can only be used with the aid of Cryoprotectors as single enzyme solutions can be stored stably at -20 ° C. Glycerin is usually used in a concentration of at least 50% Excipient that 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 cannot do cryoprotectors can 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% also reduce the storage stability of the enzymes at -20 ° C.

Surprisingly, since the appearance of the first releases on cell-free Protein biosynthesis and post-translational modification did little in the mid-1970s Technology for the production and preservation of enzymatically active cell extracts changed. In principle, there is the alternative to the already described prior art Storage stabilization by freeze drying. This method is mainly used for stabilization  of liposomes and membrane fractions (6), single enzyme preparations (1; 4; 8) or of Reaction mixtures without enzyme component (16) applied, with special sugars or other polyols in combination with bivalent metal ions or surfactants (13; 14; 15) Prevent denaturation of the enzymes or membrane structures from water loss. As has been particularly effective in stabilizing biomaterials during freeze drying the natural sugar trehalose has been proven (19; 9; 10). This sugar is from some organisms, the so-called cryptobionts, enriched in the cells in extreme dryness, and secures thus the survivability of these organisms with total water loss (17; 3).

WO 95/22605 A1, which is a document with an older seniority according to § 3.2 PatG the stabilization of proteins of the blood plasma by sugar, serum albumin and / or Gelatin and potassium salts.

WO 93/00807 A1 describes a method for stabilizing biomaterial Lyophilization using two additives, namely a cryoprotectant such. B. PEG and a sugar, known.

Describe WO 93/00807 A2, EP 0 365 685 A1 and US 5 250 429 Freeze-drying processes or freeze-dried products in which carbohydrates may can be used with other additives for stabilization.

Methods for the production of stable, complex multienyzmatic reaction mixtures, the most sensitive and complex reaction systems in molecular biology cannot be found in these documents, which are only Describe methods for long-term stabilization of proteins or protein mixtures.

The object on which the present invention is based is 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 10. The subclaims relate to special embodiments of the inventive method.

The present invention differs from the prior art for using 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 partial mixtures without enzymatic components can be prepared in a stable manner and ready for use. Secondly, conditions were found under which the trehalose guarantees sufficient storage stability without additional auxiliaries and additionally increases the reactivity of the multi-enzymatic reaction mixture 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 poreins from 5 μl of the translation mixture and qualitatively by means of 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 radioactively labeled proteins that could be precipitated by TCA could be detected (Fig. 1 / B.). This effect was based on the non-specific binding of the [ ³⁵ S] L-methionine used for the radioactive labeling to the denatured poreins.

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, that means that 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 quick-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 & 3B).

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 with 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 ul 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 ready-to-use preparation of native and artificial enzymatically active protein mixtures with reaction buffers, Cofactors and substrates.

According to the invention, the native or artificial enzymatically active protein mixtures with all the reaction components necessary for their use, the enzymatic and non-enzymatic cofactors, enzyme substrates, nucleotides and nucleosides or their oligomers, proteins, peptides, thiol compounds, RNA, DNA and derivatives of these substances, individually or in combination are, and combined with a stabilizing agent, which is a chemically inert sugar or a sugar mixture, which or in the dried state forms an amorphous glass mass with a residual water content of ≦ 0.2 g H ₂ O / g dry mass, in aqueous solution, the Concentration of the stabilizing agent in the mixture in aqueous solution is between ≧ 5% by weight and ≦ 10% by weight. They were then subjected to vacuum drying.

If necessary, there is then an overlay with inert gas or mineral oil.

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 optionally nucleic acids used.

The complex multi-enzymatic reaction mixtures produced according to the invention have the advantage that they can be stored and transported stably at 0-10 ° C. or, if appropriate, after being overlaid with an inert gas at ambient temperature (20-30 ° C.). This eliminates the technically and financially high expenditure for the purchase and maintenance of freezer equipment, as is necessary according to the conventional state of the art. Their second decisive advantage is that they are prepared ready for use with all the necessary reaction components, so that the user can only start the desired reaction by adding one, a maximum of two, key components. This enables the disadvantages of the prior art to be circumvented with regard to:

• - the simultaneous execution of a large number of parallel experiments,
• - high reproducibility of the enzymatic reactions,
• - a minimal expenditure of time for the test preparation,
• - 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.

Design example: 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

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

• 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 be absent, depending on which of the Amino acids are used to label the translation product.);
• c) 16 µl translation buffer, consisting of 120 mM K acetate, 5 mM Mg acetate and 30 wt. % 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 RNase inhibitor (40-200 U);
• h) 16 ul wheat germ extract, consisting of wheat germ lysate with a concentration of 90 A ₂₈₀ / 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 µ 30 µl can be frozen without prior shock 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 stably stored. 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 them 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 [ ³⁵ S] 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 3MM 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 (13)

1. A process for the preparation of complex multi-enzymatic storage-stable reaction mixtures for use in biochemical or molecular biological processes, characterized in that
native or artificial enzymatically active protein mixtures
with all reaction components necessary for their use, which are enzymatic and non-enzymatic cofactors, enzyme substrates, nucleotides and nucleosides or their oligomers, proteins, peptides, thiol compounds, RNA, DNA as well as derivatives of these substances individually or in combination, and
with a stabilizing agent, which is a chemically inert sugar or a sugar mixture, which, when dried, forms an amorphous glass mass with a residual water content of ≦ 0.2 g H ₂ O / g dry mass,
combined in aqueous solution, the concentration of the stabilizing agent in the mixture in aqueous solution being between ≧ 5% by weight and ≦ 10% by weight, and then subjected to vacuum drying. 2. The method according to claim 1, characterized in that native enzymatically active protein mixtures are cell extracts or fractions from these. 3. The method according to claim 1, characterized in that artificial enzymatically active protein mixtures a mixture of pre-cleaned Individual enzymes, cofactors and optionally structural proteins. 4. The method according to claim 1, characterized in that the stabilizing agent is trehalose and the vacuum drying at the same time Centrifugation takes place. 5. The method according to any one of claims 1 to 4, characterized in that the vacuum drying is carried out in a commercially available vacuum centrifuge at 30 ° C to a residual water content of 0.2 g H ₂ O / g, but at least 2.5 hours. 6. The method according to any one of claims 1 to 5, characterized in that Mix with a volume of ≧ 30 µl before vacuum drying.   7. The method according to claim 6, characterized in that freezing at -50 ° C in an alcohol / dry ice bath or in liquid nitrogen -80 ° C. 8. The method according to any one of claims 1 to 7, characterized in that the mixtures immediately after vacuum drying with an inert gas or mineral oil are coated. 9. The method according to claim 8, characterized in that nitrogen, helium or argon can be used as inert gases. 10. Use of the complex multi-enzymatic storage-stable reaction mixtures according to one of claims 1 to 9 for the synthesis, modification or analysis of Polypeptides or nucleic acids after reconstitution in water, one or each several specific key components can be added. 11. Use according to claim 10, characterized in that the key components of radioactive or non-radioactive labeled amino acids or their corresponding aminoacylated tRNA molecules, radioactive or non-radioactive labeled nucleotides or their derivatives or oligomers, natural or artificial Messenger RNA, DNA of various origins, or combinations of the above Are substances. 12. Use according to claim 10 or 11 for cell-free ribosomal protein biosynthesis and for the post-translational modification of peptides, polypeptides and proteins. 13. Use according to claim 10 or 11 for replication, reverse or non-reverse Transcription, enrichment or modification of nucleic acids.