DE19807608A1 - Measuring level of protein biosynthesis using photoprotein loaded with co-factor during translation as reporter for screening for biologically active compounds - Google Patents

Abstract

A method for monitoring the level of protein biosynthesis is new and comprises using a DNA or RNA template (I) encoding a photoprotein (II), as reporter, which is expressed in a protein biosynthesis system. (II) becomes loaded with a co-factor (III) during its translation and bioluminescence of the loaded (II) is measured.

Description

Areas of application of the method are the replacement of animal experiments and the replacement radioactive substances in pharmacology, screening of bioactive substances, Biosensors, molecular biological research, protein engineering, as well as the control of Protein biosynthesis in protein production. Furthermore, the procedure for the Determination of bacterial contamination in cases where the bacterial Pathogen on the protein biosynthesis inhibiting substances produced, for. B. Diphtheria toxin, usable.

Scientists and industry have been using the ribosomal protein biosynthesis complex, both in cell-free and in native form, as a very effective instrument for studying the translation processes and biosynthesis of proteins in analytical and preparative amounts [1, 11, 12]. Of particular interest is the possibility of synthesizing the artificial proteins (unprotected against proteases) or labeled at certain points in the cell-free system. The following methods have been used to control the protein biosynthesis process and to evaluate the results:

• - adding the translation mixture of radioactive amino acids;
• - addition of the biotinylated Lys-t-RNA;
• - parallel synthesis of proteins with enzymatic activity;
• - parallel synthesis of luciferases.

The most widely used radioactive label [2] is polluting and only gives integrated values that refer to the entire polypeptide pool. The accuracy of this method lies e.g. B. when using ¹⁴ C-Leu at about 10% in the integrated representation. Changes within 30% after 1-1.5 hours of translation cannot be registered quantitatively.

The addition of biotinylated Lys-t RNA was used to replace the radioactive label proposed [3]. This method only provides the researcher with the integral values without Difference whether the full protein or only a part was synthesized. The accuracy lies such as in the area of accuracy in radioactive labeling.

Although the synthesis of an enzyme, including luciferases, only provides information about complete biologically active protein molecules in cell-free systems [4] or cells (5), it has the same disadvantage as radioactive labeling - it only brings integrated values and to the scientist The next problem is that the enzymatic activities are very dependent on the reaction conditions. For this reason, such systems are very difficult to calibrate qualitatively. In addition, such an internal marker must meet the following conditions:

• - The substrate, product and enzyme itself must not be the protein biosynthesis system influence;
• - The polypeptide chain of the enzyme has to move in relatively quickly under synthesis conditions fold active protein;
• - since the enzymatic marker in a relatively smaller amount, in relation to the main product, is synthesized, the enzyme must have a very high specific activity;

Virtually none of the known enzymes meets all of these conditions.

As candidates for an internal reporter, the so-called Photoproteins of the coelenterate Group of special interest. These relatively small (21 kDa) proteins bind during loading the cofactor coelenterazine and oxygen. These proteins include in their Amino acid chain 2 to 3 sections, which have the property of some ions, e.g. B. Ca⁺⁺ [6] or Mn⁺⁺ ions [7] (also called ion activators) to bind selectively. Such Sections are also known for other groups of proteins and have a common one Term "EF hand". When adding ion activators, these combine with the corresponding sections and thus cause the folding of the loaded photoprotein into a compact spatial structure and the radiation of a photon.

The first protein in this group, aequorin, was released in 1962 by the research group from Dr. Shimomura (USA) isolated and characterized from sea jellyfish (Aequoria victoria) [8]. Another protein in this group, obelin, was derived from marine polyps (Obelia longissima) by a Russian research group led by Dr. Vysotsky isolated in 1988 and also characterized [9]. In addition, other photoproteins are the same Mechanisms of bioluminescence known: clytin, renillin, thalassicolin, vorin, etc.

All photoproteins in this group have a very high degree of homology in the ion activator binding areas and less similarity in the presumably binding coelenterazine Sections. The duration of the ion activator binding and folding is within one second, the binding of the coelenterazines runs much slower, the halftime is dependent on them Structure, between 2 and 4 hours.

One of the most important properties of these proteins, in contrast to luciferases, is one of them their ability in a complex with coelenterazine, oxygen and ion activators to emit a maximum of only one photon per molecule within a short time (approx. 1 second). Around to repeat this process, it is necessary the coelenteramine and the Remove ion activators from the reaction mixture and re-use the photoprotein To load coelenterazines and oxygen.  

The first attempt to monitor cell-free protein biosynthesis using a photoprotein was published in the scientific literature [10]. The procedure described includes the following steps:

• - Synthesis of apo-obelin in the cell-free protein biosynthesis system;
• - regular taking of samples from the synthesis mixture;
• - Add the Ca⁺⁺ chelator EDTA to the samples;
• - loading of the apo obelin in the samples with their cofactor coelenterazine;
• - Measuring the flash initiated by the addition of the excess of Ca⁺⁺ ions Bioluminescence.

One of the main disadvantages of this method is the fact that for loading the According to the authors, photoproteins take around 4 hours. This excludes direct control and steering through feedback in the direction Measurement data - system parameters of the biosynthesis process completely.

In addition, the known method only provides information about the total amount of photoprotein (integral value) synthesized at a particular point in time and therefore has the same disadvantage as the other types of reporter labeling described above:
the increase in the measurement signal caused by the newly synthesized protein / reporter is already after 60-90 min in comparison with the measurement signal from the previously synthesized protein / reporter. so low after the start of the process that no defined statement about the protein biosynthesis intensity is possible.

The present invention provides a method which overcomes the disadvantages of State of the art in determining the actual intensity of each Avoids timing of the protein biosynthesis process and the feedback in the direction Measurement data control enabled.

The present invention differs from the prior art for using the reporter label for protein biosynthesis control in that the photoprotein used as an internal reporter during the biosynthesis of ribosomes with a cofactor, e.g. B. Coelenterazine is loaded. The loading takes place practically simultaneously with the structure and folding of the polypeptide chains on the ribosomes. The already known (1a) and the inventive method (1b, c) are shown schematically in Fig. 1. Biolumunescence can be measured in two ways:
continuously directly in translation system or in separate samples as an integral value.

It was experimentally proven ( Fig. 2, translation of 0.1 µg obelin-m-RNA) that in the presence of the ion activator, the bioluminescence after only 10 min. starts from the start of the process. The ribosome also needs approximately this much time to synthesize the full photoprotein chain.

Therefore, our method excludes the relatively long charging time of the photoprotein and enables the immediate continuous measurement of the bioluminescence directly in the biosynthesis system in the presence of the ion activator (variant I, Fig. 1b).

The second variant, the point measurement in the total volume or in separate samples, enables such information to be obtained by adding an ion activator (variant II, Fig. 1c), although with approx. 1 min. Delay, even in cases where the ion activator can influence the translation process (e.g. living cells).

The curves shown in Fig. 2 show the incorporation of radioactive leucines in the photoprotein obelin or dehydropholate reductase, as well as the continuous bioluminescence during the translation of the photoprotein m-RNA separately or in a mixture with m-RNA encoding dehydropholate reductase. The course of the curves and the quantitative comparison show that the biosynthesis of both proteins takes place independently in one system and that the method according to the invention can be used to control the total protein biosynthesis intensity.

Since each molecule of the photoprotein can only radiate one photon under the given conditions, measuring the bioluminescence according to the first variant, directly in the protein biosynthesis system, does not give the information about the integral amounts of the synthesized product, but about the speed of protein biosynthesis at every moment ( Fig. 3).

The data recorded in variant I. or II. Enable the parameters to be adjusted immediately of the biosynthesis process.

Furthermore, in contrast to the known methods [10], the method according to the invention enables the screening of the biosynthesis-inhibiting, for. B. antibiotics, or stimulate the substances. Fig. 3a shows the inhibitory influence of the antibiotic puromycin on the protein biosynthesis system. The bioluminescence of the photoprotein was measured directly during the biosynthesis (variant I). Fig. 4 shows the dependence of the bioluminescence signal on the concentration of the two different antibiotics - cycloheximide and puromycine. In this case, the measurement was carried out according to variant II.

Since the biosynthesis of the photoproteins, like that of other proteins, by the classic way "one or more proteins coding m-RNA - ribosomal apparatus" expires, m-RNS with different origins can be used. There are, According to the current state of the art, the following sources of m-RNS are known: Transcription (in vivo and in vitro) in uncoupled or in translation Form, replication-coupled or non-coupled replication of the m-RNA, PCR technique in combination with transcription and translation or chemical synthesis. The important thing here is, that the m-RNA encoding the photoprotein internally, in the reaction mixture, or externally synthesized and the working ribosomal protein biosynthetic apparatus, both in the living eukaryotic or procaryotic cells, as well as the organelles, thermophilic  Cells, modified cells or organelles, cell-free containing protein biosynthesis system Lysates or artificial multi-enzymatic systems is made available. In all The method according to the invention can be used in these cases. That is why this relates Methods on the ribosomal translation systems and on the systems in which the ribosomal translation process with transcription, replication or with PCR / transcription is coupled. The non-tribosomal peptide or protein biosynthesis systems are off excluded the patent claims.

The temperature of the translation systems with coelenterazines remains the same as in all cases whether the translation would have been carried out without coelenterazines.

The invention is limited only to the photoproteins of the coelenterate group, which Bind coelenterazines or the cofactors with the chemical structure with the same effect and with the participation of oxygen and ion activators, the photons can radiate.

The range of ion activators is limited to the ions that deal with the E-F regions of the bind loaded photoproteins and thus cause photon radiation. As Examples are Ca⁺⁺ and Mn⁺⁺ ions.

The exemplary embodiments under P.3. show that in the case of the protoprotein cofactor maximum applicable concentration is up to 5 mM, and for the ion activator up to 1 mM are applicable. The lowest concentrations are due to the binding constants of the Cofactors with photoprotein and E-F section determined with the ion activator. For the ion The activator has a minimum concentration of 1 µM and for the cofactor - 0.05 µM.

Embodiments 1. Continuous control of translation of photoprotein in a translation system the base of wheat germ lysate according to variant I 1.1 Production of a translation mixture from individual components

Immediately before starting the experiment, the ready-to-use translation mixture of the following components is pipetted together on an ice bath:

• a) 8 µl master mix, consisting of 312.5 mM HEPES-KOH pH 7.6, 12.5 mM ATP, 1.25 mM GTP, 32 mM creatine phosphate, and 25 mM DTT.
• b) 4 µl amino acid mix consisting of 2 mM of 20 natural amino acids. The unlabeled Leu can be replaced by an equimolar amount of ¹⁴ C-Leu if necessary.
• c) 4 µl 1.0 M K-acetate and 2 µl 25 mM Mg-acetate.
• d) 4 µl creatine phosphokinase (350 U / ml).
• e) 4 µl 1.25 mM solution of the coelenterazines in methanol.
• f) 4 ul 35 mM 2-mercaptoethanol.  
• g) 32 µl wheat germ extract with a concentration of 100 A ₂₈₀ / ml.
• h) 30 µl deionized water.

The final volume of the finished translation mixture is 92 µl.

1.2 Continuous control of translation

23 µl of the translation mixture according to P.1.1 is placed in the cuvette of the luminometer. registered. After the temperature has stabilized at 26 ° C, 1 µg Obelin m-RNS in 1 µl is placed in the cuvette aqueous solution, and 1 ul of 1.0 mM Ca-acetate added and the measurement of the bio luminescence started. The measurement is in single-photon measurement mode with the accumulation time of 1 min. carried out.

The results are shown in Fig. 2 as a function of the bioluminescence signal (RLU) on the synthesis time.

2. Continuous control of translation of photoprotein in a translation system the basis of bacterial lysate according to variant I 2.1 Preparation of a translation mixture from individual components

Immediately before starting the experiment, the ready-to-use translation mixture of the following components is pipetted together on an ice bath:

• a) 8 µl master mix, consisting of 270 mM TRIS-acetate pH 8.2, 6.5 mM ATP, 5.4 mM GTP, 0.07 mg / ml folic acid, and 12 mM DTT.
• b) 15 µl amino acid mix consisting of 1.7 mM of 20 natural amino acids. The unlabeled Leu can be replaced by an equimolar amount of ¹⁴ C-Leu if necessary.
• c) 5 µl 2.0 M K-acetate and 1.5 µl 1.0 M Mg-acetate.
• d) 16 ul 250 mM acetyl phosphate.
• e) 4 µl 1.25 mM solution of the coelenterazines in methanol.
• f) 4 ul 35 mM 2-mercaptoethanol.
• g) 18 µl of S30 Esherichia coli extract.
• h) 20.5 µl deionized water.

The final volume of the finished translation mixture is 92 µl.

2.2 Continuous control of translation

23 µl of the translation mixture according to P.2.1 is placed in the cuvette of the luminometer. registered. After the temperature has stabilized at 30 ° C, 0.1 µg Obelin m-RNS in 1 µl of aqueous solution and 1 µl of 1.0 mM Ca acetate added and the measurement of  Bioluminescence started. The measurement was carried out in the single-photon measurement mode with the Accumulation time of 1 min. carried out.

3. Dependence of the translation of the photoprotein m-RNA on the concentration of the Photoprotein cofactors (coelenterazines) and ion activators (Ca⁺⁺) 3.1 Dependence on the protein cofactor

From the according to P.1.1. prepared translation mixture with ¹⁴ C-Leu and without coelenterazines were five, each. 23 µl, samples taken and tempered at 30 ° C. After the temperature has stabilized, the samples each. 1 µl or differently diluted solution of coelenterazines to the final concentrations of 0.5, 5, 50, 500, 5000 µM is added and the translation is started by adding 1 µg Obelin m-RNS in 1 µl aqueous solution per sample. After 3 hours, a 5 μl aliquot is taken from each sample and pipetted into 5 ml of 5.0% aqueous solution of trichloroacetic acid. After 10 minutes of incubation at 4 ° C., the resulting solution is filtered through GF / C glass fiber filters and the filter is rinsed with 15 ml of 5.0% trichloroacetic acid. Finally, the filter is air-dried. The radioactivity of the precipitate remaining on the filter is counted in the liquid scintillator.

The following results were registered:

3.2 Dependence on the ion activator

From the according to P.1.1. prepared translation mixture with ¹⁴ C-Leu are five, each. 23 µl, samples taken and tempered at 30 ° C. After the temperature has stabilized, the samples each. 1 µl water or differently diluted solution of Ca-acetate is added to the final concentrations of 0.0, 0.4, 4, 40, 1000 µM and the translation is started by adding 1 µl Obelin m-RNS in 1 µl aqueous solution per sample. After 3 hours, a 5 μl aliquot is taken from each sample and pipetted into 5 ml of 5.0% aqueous solution of trichloroacetic acid. After 10 minutes of incubation at 4 ° C., the resulting solution is filtered through GF / C glass fiber filters and the filter is rinsed with 15 ml of 5.0% trichloroacetic acid. Finally, the filter is air-dried. The radioactivity of the precipitate remaining on the filter is counted in the liquid scintillator.

The following results were registered:

4. Continuous measurement of translation inhibition by antibiotics

23 µl of the translation mixture according to P.1.1 is placed in the cuvette of the luminometer. registered. After the temperature has stabilized at 26 ° C, 1 µg Obelin m-RNS in 1 µl is placed in the cuvette aqueous solution, and 1 ul of 1.0 mM Ca acetate added and the measurement of Bioluminescence started. The measurement is carried out in the single-photon measurement mode with a Accumulation time of 1 min. carried out. After 50 min. the translation system 1 µl 2.5 mM Puromycine added and the bioluminescence signal registered further.

The results are shown in Fig. 3 as a function of the bioluminescence signal (RLU) on the synthesis time.

5. Measurement of the protein biosynthesis effectiveness according to variant II

From the according to P.1.1. prepared translation mixture with ¹⁴ C-Leu and without coelenterazines are 16, each. 23 µl, samples taken and tempered at 26 ° C. After the temperature has stabilized, the samples each. 1 µl of the differently diluted solution of Puromycine or Cycloheximide to the final concentration of 0.036 0.39, 0.78, 1.56, 3.12, 6.25, 12.5 and 100 µM is added and the translation is made by adding 1 µg Obelin m-RNA in 1 µl aqueous solution per Rehearsal started. After 3 hours of translation, 10 µl aliquots are taken from each sample, with 90 µl of the loading solution consisting of 10 mM Tris-HCl pH 7.1, 50 mM NaCl, 1 mM EDTA, 0.1% gelatin, 140 mM 2-mercaptoethanol and 0.4 µM Coelenterazines, diluted and placed in the cuvette of the luminometer. The flash luminescence is started by adding 100 µl 10 mM Ca-Acetete solution and as an integral value within the first 2 min. registered.

The same integral values are obtained when Ca ions are added to the final concentration from 1 µM to 1 M registered.

The results are shown in Fig. 4 as a function of the integral values of the bioluminescence signal on the concentration of the antibiotics.

Bibliography

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drawings

Fig. 1. Representation of the known methods (1a) and methods according to the invention. Bioluminescence is measured in two ways - continuously (1b) or in separate samples (1c).

Fig. 2. Continuous measurement of bioluminescence (RLU) during the biosynthesis of the photoprotein obelin separately or together with the dehydropholate reduction. In order to demonstrate the actual translation of both m-RNAs, ¹⁴ C-Leu was added to the radioactive marker system.

Fig. 3. Inhibition of protein biosynthesis by adding the antibiotic puromycin.

Fig. 4. Dependence of the integral values of the bioluminescence signal on the concentration of the antibiotics.

Claims (13)

1. A method for controlling the protein biosynthesis intensity by means of expression of a reporter protein using an RNA or DNA template and measuring its activity, characterized in that the DNA or m-RNA template corresponds to a photoprotein, the expression is carried out in a protein biosynthesis system and the photoprotein is loaded with its cofactor during translation and the bioluminescence of the loaded photoprotein is measured. 2. The method according to claim 1, characterized in that the template of the photoprotein as an expressible single-protein DNA or m-RNA construction or as part of a complex DNA or m-RNA construction is used. 3. The method according to claim 1 and 2, characterized in that the photoprotein m-RNA into the translation system as a finished construction directly or by means of transfection is added, or which forms directly in the translation volume by means of the Translation coupled transcription, RNA polymerization, replication or PCR technique. 4. The method according to claim 1, characterized in that the photoprotein, for. B. apo- Obelin, has a natural or artificially constructed order of the amino acids and for Coelenterate group belongs. 5. The method according to claim 1, characterized in that the photoprotein cofactor Coelenterazin structure or chemical structure having the same effect. 6. The method according to claim 1, characterized in that the protein biosynthesis system a native eukaryotic, procaryotic, thermophilic, or an artificial one set multi-enzymatic cell-free protein biosynthesis system. 7. The method according to claim 1, characterized in that the protein biosynthesis system a component of the native or modified eukaryotic, procaryotic, thermophilic Cells or organelles. 8. The method according to claim 1 to 6, characterized in that the bioluminescence continuously, in the presence of an ion activator, e.g. B. Ca⁺⁺ ions during biosynthesis or as a point measurement in the total volume or in the separate samples by adding of an ion activator is measured. 9. The method according to claim 1 to 5 and 7, characterized in that the bioluminescence of the loaded photoprotein separately from the cells or organelles by adding the Ion activator is measured. 10. The method according to claim 1, 8 and 9, characterized in that the concentration of Ions activator after the addition in the biosynthesis system or in the separate samples in the Range is from 1 µM to 1 mM.   11. The method according to claim 8 to 10, characterized in that the ion activator is an ion which binds to the E-F region in the photoprotein and thus the bioluminescence of the Causes photoproteins. 12. The method according to claim 1 and 8, characterized in that the ion activator in the Samples are added until a concentration in the range of 1 µM to 1 M is reached. 13. The method according to claim 1 and 4, characterized in that the concentration of Photoprotein cofactors in the reaction mixture are in the range from 0.05 µM to 5 mM.