The method of synthesizing a fluorescently-labeled protein

 

(57) Abstract:

The invention can be used in biotechnology. In a cell-free system for protein synthesis in incubated samples of ribosomes with plasmid DNA coding necessary protein. Incubation is carried out in the environment for coupled transcription/translation together with the aminoacyl-tRNA with a fluorescent tag. Then the purified protein, measure its amount, and detect fluorescence and biological activity. Application of the fluorescent label on the N-end of the synthesized protein can be reliably diagnose synthesized proteins that retain full enzymatic activity. 8 C.p. f-crystals, 6 tab., 4 Il.

The invention relates in General to the fields of molecular biology, biotechnology and protein synthesis. More specifically, this invention relates to a new method of synthesis of a fluorescently labeled protein in a cell-free system for protein synthesis.

Prior art

A number of indications suggest that nascent (emerging) polypeptides acquire secondary and tertiary structure on the ribosome. Beckmann and his associates (1990) found that Hsp70 interacts with nascent proteins and postulates hypothesis that the growing peptide can be synthesized in the form of an alpha helix. Reviewed the application of fluorescent methods to study the elongation and laying the growing peptides and proteins on ribosomes. Previously, these methods were used to demonstrate that increasing protein MS2 fit, being associated with ribosomes. It was shown that the so-called "mentors" (factors involved in ensuring the Assembly of the spatial structure of proteins - author, translator) facilitate the laying of many proteins from their denatured state, but very little is known about function whether these proteins on ribosomes during translation. Hartl and colleagues have presented evidence suggesting that DnaJ is the first "mentor", which is associated with the growing peptides luciferase Firefly or chloramphenicolchloramphenicol on ribosomes wheat germ (Hendrick et al., 1993).

In prior knowledge was not an effective means of synthesis of fluorescently labeled protein in the cell-free system for protein synthesis. This invention solves this problem.

Disclosure of the invention

In this invention the coumarin derivatives of coumarin-maleimide-SAcMet tRNA (CPM-SAcMet-f) included in the N-end growing is reply/broadcast. Methods using fluorescence was used to monitor changes in the local environment and the mobility of the N-terminal probe and turn actions mentors to the installation, activation, and selection is growing (nascent) or full-length polypeptide from the ribosome. This invention shows that mentors affect the laying of the growing polypeptide Rodney at a time when they are connected in the form peptidyl-tRNA on the ribosome. Mentors facilitate their release from the ribosome in the form of enzymatically active protein.

In one embodiment of the present invention is provided a method of synthesizing a fluorescently labeled protein in a cell-free system for protein synthesis, which includes the following stages: (a) incubating the sample of ribosomes obtained from cell-free extract with plasmid DNA containing the coding sequence for the target protein, and the sample incubated in the medium for coupled transcription/translation together with the aminoacyl-tRNA with a fluorescent label; (b) partial purification of fluorescently labeled protein by separating newly synthesized fluorescently labeled protein from other fluorescent components within the sample; (C) measuring the amount of sicoi activity of the newly synthesized protein.

Other and further aspects, features and advantages of this invention will be apparent from the following description of preferred at present variants of the invention, given for the purpose of describing.

Brief description of drawings

For a clearer understanding of the above features, advantages and objectives of the present invention presents a more detailed description of the invention with references to some of its variants, which are illustrated by the accompanying drawings. These drawings form part of this application. However, it should be noted that the accompanying drawings illustrate the preferred variants of the invention and therefore should not be construed as limiting the invention in their volume.

Fig.1 shows the analysis of free and bound with ribosomes Rodney or translated in the presence of [14C]-leucine or initiated CPM-Sac-[35S]-Met-f. Rhodanese synthesized by coupled transcription/translation or with [14C] -leucine (160 CI/mol) or CPM-Sac-[35S]-Met-f (4000 CI/mol) as the radioactive precursor. After incubation, the reaction mixture was centrifuged. Supernatant and resuspendable the first 2 parts, labeled14C-leucine and CPM [35S] -Met, respectively). Lanes 1 and 2 - incubation with internal [14C] -leucine; lanes 3 and 4 - incubation with N-terminal CPM - Sac-[35S]-methionine. Lanes 1 and 3 to 20 μl of the supernatant, lanes 2 and 4 to 20 ál resuspending ribosomes. The third part of Fig. 1 (labeled antibodies against RHO) shows the Western blot, probed with antibodies against Rodney. The position of horseradish peroxidase covalently associated with the second antibody was visualized using the ECL system Amersham. The arrow indicates the electrophoretic mobility of native rhodanese from bovine liver.

Fig. 2 shows the fluorescence quenching of coumarin nitromethane. The graphs of the Stern - Volmer fluorescence intensity steady state for CPM - rhodanese released from ribosomes or associated with ribosomes, in comparison with free CPM-Sac-Met-tRNA (line 1) and this tRNA associated with ribosomes (line 6). Line 2 - CPM-rhodanese in the supernatant in enzymatically active form; line 3 - CPM-rhodanese released with the help of Dnak line 4 - CPM-rhodanese released by puromycin; line 5-CPM-rhodanese associated with ribosomes.

Fig. 3 shows emission spectra of fluorescence CPM-maceneaney with ribosomes (A) or isolated in the supernatant (B) (solid line). Then added IgG against coumarin and again shot spectra (dotted line). The insert provides quantitative data of fluorescence for the respective analyzed samples.

Fig. 4 shows that DnaJ affects the interaction of IgG against coumarin with associated with ribosomes CPM-rodanthe. First shot range CPM-rhodanese on ribosomes after incubation with sparsomycin (solid line). Established three parallel samples (CPM-rhodanese on ribosomes incubated with sparsomycin), to which was added DnaJ or DnaK separately or together. Shot spectra (not shown) and analyzed. It was found that these data are identical or very similar to the data shown in table VI. Then each sample was added IgG against coumarin. After incubation for 10 minutes at room temperature filmed spectra, which are presented here. (x-x)DnaJ+IgG; (...)DnaK+IgG; (- -)DnaJ and DnaK+IgG.

Detailed description of the invention

In the description of the present invention uses the following abbreviations: RHO-rhodanese; CPM-3-(4-maleimidomethyl)-4-methyl-7- (diethylamino)-coumarin; CPM-rhodanese-rhodanese, labeled at its N-terminal of the methionine CPM; SDS-PAGE gel electrophoresis in polyacrylamide gel with dodecylsulfate the RNA, which was aminoacridone methionine and then marked on the amino group of methionine through mercaptohexanol acid using CPM; IgG immunoglobulin; Fab - it generated by papain fragment.

This invention relates to a method of synthesizing a fluorescently labeled protein in a cell-free system for protein synthesis, which includes the following stages: (a) incubating the sample of ribosomes obtained from cell-free extract with plasmid DNA containing the coding sequence for the protein of interest, and the sample incubated in the medium for coupled transcription/translation together with the amino-acyl-tRNA with fluorescent accurate; (b) partial purification of fluorescently labeled protein by separating newly synthesized fluorescently labeled protein from other fluorescent components within the sample; (C) measuring the amount of synthesized protein; (d) determination of the fluorescence of the newly synthesized protein and (e) determining the biological activity of the newly synthesized protein.

Usually, in this invention, the fluorescent label is located on the N-end of the protein. In the preferred embodiment, used in this way aminoacyl-tRNA is the, the initiator tRNA.

In the method of the present invention, the environment for transcription/translation usually contains RNA polymerase, nucleosidases, a system for regenerating energy and amino acids in a buffered saline solution. In a preferred embodiment, ribosomes isolated from an extract of E. coli, called S30. In a preferred embodiment, the plasmid DNA is linear. Preferably, the incubation stage (a) is conducted for approximately 30 minutes at approximately 37oC. in the method of the present invention fluorescently labeled protein is partially purified by a method selected from the group consisting of centrifugation, ion exchange chromatography and gel filtration.

The following examples are given to illustrate different variants of the invention and are not intended for any limitation of the present invention.

Example 1

The chemicals.

Nucleosidase and E. coli tRNA were supplied by Boehringer-Mannheim; 3-(4-maleimidomethyl)-7-diethylamino-4-methylcoumarin (CPM) from Molecular Probes Jnc. (bugene, OR). tRNAMetf rifampicin, puromycin, sparsomycin and all other reagents were supplied Sigma. A mixture of ribonuclease A and TI ("Misny cocktail") was from Ambion (Austin. TX). [14C]-boat containing the gene Rodney plasmid, rhodanese isolated from mitochondria bovine liver, and polyclonal antibodies against Rodney were obtained from Dr. Paul Horowitz (University of Texas Health Science Cenrte, San Antonio, TX). Polyclonal antibodies against factor release (releasing factors) E. coli 1 and 2 were provided by Dr. Warren Tate (Universitu of Otago, New Zealand). The second antibody (goat antibodies against rabbit IgG-horseradish peroxidase and rabbit antibodies against sheep IgG-horseradish peroxidase were purchased from South San Francisco, CA).

Example 2

In vitro systems

Education plasmids, selection of RNA polymerase SP6 and obtaining cell-free extract of E. coli (S30), and the allocation of a fraction of ribosomes from S30 performed as described Kudlicki et al., Anal. Biochem. 206:389 (1992). Basically, plasmid preparations were prepared according to standard procedures (J. Sambrook et al. , (1989) Molecular Cloning, Cold Spring Harbor Zaboratory Press), except that the stage of centrifugation in Cs Cl was replaced Q Cartridge chromatography (Bio-Rad). RNA polymerase SP6 and T7 commercially available. The applicable fraction of ribosomes were separated from the S30 fraction obtained according to method G. Zubay from E. coli K12 (A19). Cells were grown at 37oC in LB (Sigma) broth, to which was added 20% glucose (10 ml per 2 litres of medium). Cells were harvested in mid-logarithmic phase R Xg and received the S30 extract. To the buffer for lysis added protease inhibitor, phenylmethylsulfonyl, to a final concentration of 0.5 mm. Aliquots S30 (9 ml each) were centrifuged at 47000 rpm, for 4 hours in the Ti rotor 50 Beckman. Precipitated ribosomes resuspendable in 1.0 ml of 20 mm Tris-HCl (pH 7.5), 10 mm Mg (OAc)2and 1 mm DTT, getting the suspension to approximately 1200 A260units/ml of This fraction of ribosomes were stored at -70oC in the form of a small aliquot.

The drug CPM-SAcMet-f used, as described previously, for marking-amino group of Phe-tRNAPheCPM (Odom et al., 1990). Briefly, the amino group of methionine [35S]-Met-f was etilirovany dithiodipropionic acid and then restored to the appropriate derived thioglycolic. Its sulfhydryl group is reacted with maleimide (CPM). Antibodies against CPM received in rabbit, IgG fraction of the serum of immunized rabbit were obtained as described (Picling et al., 1992).

In vitro system for coupled transcription/translation was described in detail (Kudlicki et al., 1992). In short, the system is used for holding the coupled transcription/translation contained in a total volume of 30 μl: 50 mm Tris-acetate (pH 7.8), 14 mm Mg (OAc)2, 36 mm NH4OAc, 72 mm KOAc, 2 mm Ca(OAc)2, 0.5 mm EDTMP, 27 mm phosphoenolpyruvate, of 0.35 μg of pyruvate kinase, 1 µg folinovoy acid, 83 μm14C-leucine, 330 μm each of the other 19 amino acids, 20 μg E. coli tRNA (Boehringer) 0.5 μg rifampicin, 0.3 mm glucose-6-phosphate, 1,2-A260-units fractions of E. coli ribosomes. 0.5 μg of plasmid DNA and 0.5 μg of RNA polymerase SP6. For this test14C-leucine was diluted to 40 CI/mol. Incubation was carried out for 30 minutes at 37oC. For large-scale synthesis of CPM-rhodanese the volume of the reaction mixture increased to 0.9 ml, and they contain salts and low molecular weight components listed above, plus 5 mm Na2S2O3. Approximately 60 A260-units sepromatiq ribosomes were incubated with approximately 10-15 mcg with non-linear plasmid (SP65) containing the coding rhodanese sequence under control of the SP6 promoter, CPM-SAc[35S]-Met-f(350 CI/mol) was used as the radioactive precursor; polynovo acid not included in the mixture. In some control experiments used a [14C] -leucine (40 CI/mol) together with non-radioactive fMet-f. After incubation for 30 minutes at 37oC the sample was applied at 0.6 ml of buffered sucrose and centrifuged for 45 minutes at 45000 rpm Then collect points and the 20 mm Tris-HCl, pH 7.5, 10 mm Mg (OAc)2, 30 mm NH4OAc, 1 mm DTT, 5 mm Na2S2O3(solution A). Immediately after centrifugation the supernatant fraction (approximately 0.9 ml) was treated with ribonuclease A and T1 (0.1 mg and 2000 u/ml, respectively) for 15 minutes at 37oC for the destruction of the remaining CPM-SAc-[35S-Met-f. Then, the resulting reaction mixture was chromatographically on a column of Sephadex G100 (h cm), equilibrated with solution A, to separate the newly synthesized CPM-labeled rhodanese from low molecular weight degradation products containing coumarin and [35S]-methionine.

Example 3

Measurement of fluorescence

Measurement of fluorescence was performed by counting the photons spectrofluorometer model 8000C of SLM-Aminco Instruments Inc. (Urbana, IL). Spectra were measured at intervals of emission of 1 nm at a scan speed of 0.5 seconds per increment (increment) wavelength; the wavelength of excitation was 390 nm. Spectra and relative quantum yields were normalized on the basis of radioactivity from [35S]-methionine, which was present in the form CPM-SAc-methionine in the sample. The relative quantum yields of fluorescence was determined from the integrated emission spectra. The relative quantum yield CPM-rhodanese in ropey fluorescence was determined, as described in Odom et al., (1984), when the wavelength of emission of 480 nm. For studies of the fluorescence of the sample resuspending ribosomes or fractions of the treated supernatant were incubated in a cell in the total volume of 420 μl, containing salts and low molecular weight components described above for coupled transcription/translation, but without amino acids and without CTS and TTF (solution B). After removing the spectrum of the tested components were added in a minimal volume. Cell were incubated for 10 minutes at 37oC and then re-shot range. Additives: anti-CPM-IgG, 0.1 mg; mentors GroES, 1,6 µg; Chaperonin, 6 mcg; DnaK, 4 µg; DnaJ 2 mcg; GrpE: 3 µg.

Example 4

Determine the number Rodney

Deposition of protein trichloroacetic acid (THU) to determine the number of polypeptides Rodney, analysis by electrophoresis in SDS page-ordinator and autoradiography was performed by methods well known in this field of knowledge. Activity Rodney was determined according Sorbo (1953) in the description Tsalkova et al. (1993). Basically enzymatic activity Rodney was determined by calorimetric analysis, measuring the conversion CN-in SCN-enzyme using S2O32-as the substrate. Formed SCN-has detected valentinovo iron. One unit took the amount of enzyme that generates 1 μmol of product per minute at 37oC in this test system.

Western blots of electrophoresis in SDS page-ordinator probed bovine antibodies against factors release 1 or 2 or rabbit antibodies against Rodney, then the appropriate second antibody, which was connected to horseradish peroxidase. Peroxidase was detected by chemiluminescence using ECL products Amersham.

Example 5

Laying Rodney, coumarin labeled at its N end

To monitor the actions of "mentors" during the installation and activation of nascent (growing) Rodney coumarin translational embedded in the N-end of the enzyme from CPM-SAc[35S]-Met-f formed enzyme was derivatively chemical reaction and then used to initiate protein synthesis on ribosomes of E. coli, as described above. The inclusion of CPM-SAc[35S]-methionine at the N-ends of the polypeptides produced in the system of coupled transcription/translation, the observed radioactivity after deposition of trichloroacetic acid synthesized in vitro polypeptide Rodney. The product was identified by electrophoresis in SDS page-ordinator and autoradiography, as shown in Fig. 1. And the amide gels full rhodanese in the supernatant and in the fraction of ribosomes respectively.

To further identify these full forms of the product synthesized during coupled transcription/translation of polypeptides of the LTO-polyacrylamide gel were transferred to PVDF-membrane and probed with antibodies against Rodney. Because associated with the ribosome, the polypeptide can be transformed into an enzymatically active soluble protein, this invention clearly shows that this polypeptide is a precursor of the soluble enzyme.

The nature of the differences between soluble and associated with ribosome-polypeptide is not known. One of the proposals is that the more slowly migrating form covalently linked to tRNA or produced from it the nucleotide fragment. That this hypothesis is incorrect, it is proved by the following observations: on the nature of the electrophoretic migration is not affected preincubation samples with a mixture of ribonuclease or predisturbance at a pH of 10.5 in the 100oC for 10 minutes. The ester linkage between the peptide and the 3'-terminal ribose of tRNA would be either hydrolyzed under the above conditions. In addition, relatively slowly migrating polypeptide was not detected with the associated with the ribosomes formability applies only to rhodanese. Nature is slowly migrating form was not established, but it is most likely that it is formed by translation through the termination codon UGA, located at the end of the coding sequence Rodney that leads to broadcast thirteen additional codons prior to the onset of termination at codon UAG in phase. However, it is somewhat surprising that this longer form of the precursor to quantitatively transformed into a soluble form during activation and release of the polypeptide.

Radioactively labeled methionine from CPM-SAc[35S]-Met-f added to the reaction mixture can be placed only on the N-end Rodney, but not inside it. Thioester bond between CPM and thiol methionine, acylated mercaptohexanol acid, chemically and enzymatically stable under the applied conditions of incubation. IDA-SAcMet-tRNA is not decelerate methionyl-tRNA synthetase and tRNAMetm not able to enzymatic aminoacylase IDA SAc[35S] methionine. In addition, the reaction mixture consisted of a large excess of unlabeled methionine (350 microns). This significantly reduced the specific radioactivity of free [35S]-methionine, which could be present and to ensure the inclusion of methionine voraciousness of [35S] -methionine, and radioactivity was used to normalize the fluorescence intensity and spectral data so that the values given below for these parameters is directly proportional to the quantum yield of fluorescence can be compared between different experiments.

Example 6

The distribution of newly synthesized CPM-labeled Rodney

The distribution of newly synthesized CPM-labeled rhodanese in the soluble fraction and the fraction of ribosomes, respectively, were analyzed after deposition of ribosomes by centrifugation through a layer of sucrose solution immediately after its synthesis. From table 1 it is evident that [14C]-leucine was included in the internal regulations of the polypeptides Rodney, N-end which have not been modified coumarin. In this case, initiation of protein synthesis used non-radioactive fMet-f. Approximately half of these newly synthesized polypeptides (62% in the calculation of [35S]-methionine and 43% in the calculation of [14C]-leucine) was detected in the fraction of ribosomes. Much of the remainder of the newly formed protein was extracted from the supernatant. The protein product at a fraction of the supernatant completely was a full-sized rhodanese, like the IG. 1).

Molar quantity Rodney synthesized in a cell-free system of transcription/translation, was determined from the incorporation of [35S] -methionine or [14C-leucine into polypeptides Rodney. In case only one of the coding sequence was transcarbamoylase from this plasmid (pSP65), namely mRNA for rhodanese. The inclusion of [14C]-leucine into peptides in the reaction mixture without the plasmid was less than 5% of the inclusions in the presence of plasmids, and these data are not considered. Chain full Rodney contains 24 leucine residue and has a molecular weight of 33,000. Thus, knowing the percentage of a full-sized product (almost 100% for a fraction of the supernatant), the molar quantity of full-size protein that was synthesized, can be calculated from the incorporation of N-terminal methionine or internal leucine. This value can be used to calculate the specific activity of enzyme units/ mg rhodanese. The units of activity Rodney defined in moles of thiocyanate formed per minute.

Example 7

Activity Rodney

The specific enzymatic activity Rodney at a fraction of the supernatant was 744 units/mg Rodney relatively included [14C]-leucine. This act is camping product transmitted from the ribosome in a fraction of the supernatant was placed in a native conformation. In contrast, growing polypeptides on ribosomes did not possess enzymatic activity. A similar situation occurs during the formation of the coumarin-[35S]-methionine N-Terminus of newly synthesized Rodney: rhodanese in the supernatant active, whereas polypeptides on ribosomes enzyme inactive.

A smaller number of [35S]-methionine-labeled Rodney was extracted from ribosomes during protein synthesis with N-terminal probe, but its average specific enzymatic activity (833 units/mg rhodanese) was about 17% higher than the activity of the native enzyme. This is likely caused by the removal of the N-terminal [35S]-methionine from newly formed enzyme.

Example 8

The stability of the newly synthesized Rodney

Stability CPM-[35S]-methionine at the N-end of the newly synthesized Rodney felt directly in the supernatant and in the fraction of ribosomes, respectively. The reduction of trichloroacetic acid precipitated radioactivity of approximately 10% was observed after 60 minutes incubation at 37oC, whereas in the supernatant fraction not Balestri specific enzymes, remove the methionine from the N-Terminus of many proteins. In the case of in vitro synthesized Rodney, the polypeptide without the N-terminal coumarin-[35S]-methionine, not detected radioactivity, which led to the result, conservative amount of protein rhodanese present in these samples. Higher specific enzyme activity of the supernatant fraction, calculated from N-terminal methionine (833 units/mg compared with 744 units/mg per internal leucine, table 1), presumably reflects the underestimation of protein rhodanese present in the sample. Also, a small portion of the molecules Rodney could be initiated unlabeled endogenous formylmethionine of Met-tRNA, which were not eliminated during retrieval faction S30. It should be noted that immediately after synthesis and centrifugation the supernatant was treated with ribonuclease, then chromatographically on Sephadex G100 for the Department of CPM-labeled rhodanese from low molecular weight fluorescent compounds. In any case, table 1 indicates that coumarin may be included in the N-end rhodanese, and that these molecules are enzymatically active upon release from the ribosome.

Rhodanese synthesized coupled transcription/translation with CPM-SAc[oC the reaction mixture was centrifuged, receiving a fraction of ribosomes and the supernatant analyzed separately for the amount of synthesized protein or enzymatic activity. The number given for the reaction mixture by volume of 30 µl. The specific activity of the enzyme (native rhodanese) is equal to approximately 750 units/ml of protein,1is not expected due to the presence of partial and full-inactive Rodney on ribosomes.2-expected pmol of enzyme in brackets (24 leucine residue in the polypeptide Rodney).3this average specific enzymatic activity was always higher than the specific activity of native rhodanese.

Example 9

Characteristics of fluorescence CPM-labeled Rodney

The parameters of fluorescence CPM-SAc-Met-Rodney free in the supernatant or associated with ribosomes, are summarized in table 11. The corresponding emission spectra are given in Fig. 3A and 3B. The corresponding emission spectra are given in Fig. 3A and 3B. For comparison, in table 11 include data for CPM-SAc-Met-f free in solution or associated with ribosomes. The molar number labeled on the N-end CPM-SAcf-Met-f or rhodanese (based on [35S] is the capacity to the total molar amounts of coumarin. Quantum yield of fluorescence (Q) is given relative to the quantum yield of the enzymatic active CPM-SAcMet-rhodanese in the supernatant fraction, for which the relative quantum yield was adopted for 1,00 (table 11). Thus, it is possible to compare spectra and relative quantum yields, are presented in the drawings and tables.

Quantum yield of the enzymatic active CPM-rhodanese in the supernatant fraction was slightly higher than the quantum yield CPM-rhodanese associated with ribosomes (relative quantum yield of 1.0 against 0,89), but both were significantly higher than the fluorescence of the CPM-SAcMet-f as free in solution (Q= 0,43), and associated in the peptidyl transferase center of the ribosome (G= 0,63). The latter is the case prior to initiating peptide synthesis with the formation of nascent (growing) Rodney with CPM-SAc-methionine at its N end. This quantum yield reflects the local environment coumarin probe. For the free coumarin or CPM-cysteine quantum yield increases with bathochromic shift in emission spectrum with solvents of decreasing polarity. For comparison, the absolute quantum yield and the maximum emission for free CPM-cysteine equal 0,31 and 486 nm, sooye data of table II, apparently, indicate that coumarin probe CPM-SAcMet-f associated with the ribosome encounters a more hydrophobic environment, when it is present on the N-end of the nascent peptide, but is still held in the form peptidyl-tRNA on the ribosome. There was a further increase of the quantum yield as liberation growing rhodanese in the supernatant fraction in the form of enzymatically active form. These differences probably reflect different stages in laying the growing Rodney gradually in the native conformation, as will be discussed below. The fluorescence anisotropy was approximately 0,17 for CPM-Met-f, free in solution, and approximately 0,36 when associated with ribosomes condition (table II). Anisotropy for CPM-Met at the N-end associated with ribosomes Rodney was 0,31, but fell to 0.18 with the release of the enzyme from ribosomes in enzymatically active form in the supernatant. This difference presumably reflects the difficulty of movement of the fluorophore, which is created by the ribosome, due to steric barriers to the movement of the probe and/or the difference in the rotational relaxation or time spent in the solution of the free rhodanese and is associated with ribosomes Rodney, respectively. The last parameter reflects DNAME or identical samples used to determine the synthesis and enzymatic activity Rodney presented in table I. For comparison included data on fluorescence for CPM-SAcMet-tRNA (item B. above).1Emaxthe wavelength of the emission spectrum,2the relative. Q is the relative quantum yield of fluorescence; it was determined from the integrated emission spectrum of fluorescence, normalized on the basis of the radioactivity of [35S]-methionine and expressed relative to the quantum yield of the enzymatic active CPM-SAcMet-rhodanese (compare table 1) in the supernatant fraction. 3A - anisotropy fluorescence.

Example 10

The molecular action "mentors" associated with ribosomes CPM-rhodanese

The enzyme is released from the ribosome in the presence of "mentors" was enzymatically active with a specific activity similar to the specific activity of native rhodanese, and contained a high proportion of enzyme molecules in their native conformation. In contrast, associated with ribosomes rhodanese was enzymatically inactive.

This invention illustrates the effect of "mentors" (DnaJ, DnaK, GrpE, Chaperonin, GroES) on the fluorescence of associated with ribosomes, enzymatic inactive growing on the enzyme. The last parameter was taken as a measure of the correct installation of rhodanese in native conformation. Each of the five "mentors" were added separately, then all together at the same time to the reaction mixture containing ribosomes with a full-size rodanthe, when retranslateui conditions.

Table IIIA shows that mentors DnaK and GroES (each) cause a significant release of polypeptides Rodney, as reflected by a decrease in fluorescence anisotropy. In contrast, DnaJ gave a small but significant increase in anisotropy. Mentors GrpE and Chaperonin had very little effect on the fluorescence. Significantly greater effect on the amount of protein released from the ribosome, and the fluorescence was observed after incubation of ribosomes with all the mentors at the same time. Then the reaction mixture was separated by centrifugation at a fraction of ribosomes and supernatant.

The number in polach [35S]-methionine in the polypeptides rhodanese in the supernatant fraction is given in table IIIB. The specific enzymatic activity rhodanese in the supernatant fraction after incubation with all mentors together approached the specific activity of native rhodanese, 695 units/mg compared with 745 for NK similar to these parameters for the enzymatic active rhodanese (cf. table II). In contrast, rhodanese released from ribosomes in the presence of only DnaK or GroES, was essentially the enzyme inactive, although it consisted of more than 90% of the full-length polypeptides rhodanese (data not shown). Its quantum yield and the maximum emission differed from these parameters, enzymatic active rhodanese that suggests that this material was not placed in the native conformation. Thus, this invention demonstrates that molecular mentors reinforce effective folding into the native conformation only if their joint effect on associated with ribosomes growing rhodanese. DnaK or GroES cause some release of nascent chains from ribosomes, but other mentors are required to complete the process of laying on ribosomes.

Ribosomes containing 4.5 pmol CPM-SAc[35S]-methionine in the growing peptides Rodney, incubated for 10 minutes at 37oC when retranslateui conditions with these mentors. Characteristics of fluorescence of the reaction mixtures was determined either immediately (part a), or for a fraction of the supernatant only after centrifugation (part B). Enzymatic UP>- material unsuitable for reliable measurement of fluorescence.

Quality commercial mentors were assessed by electrophoresis in SDS page-ordinator with subsequent staining of Kumasi. Mentors gave a single band of the expected size or the expected electrophoretic mobility, except GroES, which contained a number of more high molecular weight impurities, and DnaJ, which gave dual band. All the mentors felt the presence of the releasing factor 1 or releasing factor 2 in the form of impurities. Western blots from SDS page gels-ordinator was probed with appropriate antibodies. None of the releasing factors was not detected in any of the drugs mentors using this method (data not shown).

Table III shows that no single mentor was not able to convert inactive associated with ribosomes rhodanese in natural conformation. Then examined the sequential addition of one or two mentors. An unexpected aspect of this invention, illustrated in table IV, was the fact that, apparently, it is important to add. Enzymatic active rhodanese received only in the case if you added DnaJ, but the decrease in anisotropy and an increase in the relative quantum yield. In contrast, the anisotropy remained high and quantum yield were changed very little, when Chaperonin/ES were the first mentors with whom incubated associated with ribosomes polypeptides Rodney, and then added missing mentors (section B in table IV). In this situation, the enzymatic activity was close to the lower limit of detection. In table IV parameters of fluorescence was determined in the whole of the reaction mixture, whereas the enzymatic activity was determined in the supernatant fraction after centrifugation, Chaperonin/ES can act on the fluorescent molecules Rodney still associated with ribosomes, or molecules released before adding Chaperonin/ES.

Ribosomes containing 4.5 pmol CPM-SAc[35S]-methionine growing polypeptide Rodney, incubated for 10 minutes at 37oC with these mentors in the order shown in paragraphs A or B. the Parameters of fluorescence was determined as described above, then the sample was centrifuged and the resulting supernatant analyzed for the amount released from ribosomes Rodney and enzymatic activity, which was calculated as the specific enzymatic activity,1-abbreviations listed in table II.2and is from the supernatant, as explained above.

Example 11

Requirements for the conversion of the enzyme inactive Rodney

This invention also shows the necessary conditions in respect of mentors and incubation for enzymatic conversion of inactive rhodanese in enzymatically active conformation of the enzyme. The work, presented in table IV was repeated until, when added DnaK/GrpE (before adding GrpEL+GroES) then, the sample was centrifuged for quick separation of the released protein from associated with ribosomes peptides Rodney. The supernatant containing released from ribosomes full sized peptides Rodney, pietravalle back in the cuvette for the measurement of fluorescence was determined by fluorescence parameters (table V, section A, line 1) and added

Chaperonin/ES (line 2). After 10 minutes incubation again shot range and expected anisotropy, as described above. Likewise analyzed the fraction of ribosomes containing associated with ribosomes Rodney after her resuspendable in solution B and re-incubated in the absence or in the presence of Chaperonin/ES. Then determined the spectral properties of these samples (table VB, the first 2 columns). Finally, samples again zentrifugenbau obedennoy Rodney, who was present in a fraction of the supernatant (table VB, the last 2 columns). Table V shows that approximately 1.8 pmol CPM-rhodanese (approximately 30% associated with ribosomes CPM-rhodanese in the original sample) were released in the supernatant after incubation with DnaJ, DnaK and GrpE, but without adding Chaperonin/ ES. This material had a low anisotropy (as expected) and had no enzymatic activity. Adding Chaperonin/ES to this fraction, containing released CPM-rhodanese, had no effect on the anisotropy and had only a minimal effect on enzymatic activity. CPM-rhodanese associated with ribosomes, had a very high anisotropy before adding Chaperonin/ES, because CPM-rhodanese, which is released from the ribosome with the help of DnaK during the previous incubation, were excluded from the sample by centrifugation. Anisotropy fell sharply adding Chaperonin/ES (table VB) and detected enzymatic activity. The specific enzymatic activity of released this protein was equal to the specific activity of the native enzyme.

Table V shows that the Chaperonin/ES has a minor effect or no effect on the released polypeptides Rodney, but, apparently, causes activecool with DnaK, DnaJ and GrpE. For comparison, in the VC table includes the results for polypeptides Rodney released from ribosomes by puromycin. None of the mentors had no detected no action on the enzymatic activity or fluorescence from CPM-rhodanese after incubation of these ribosomes with puromycin. Similarly, none of the mentors, separately or in combination, did not exert any action on the enzymatic activity or fluorescence enzyme active CPM-rhodanese, osvobodivsheysya of ribosomes during source coupled transcription/translation (data not shown).

Table V CPM-Met-rhodanese associated with ribosomes (approximately 4.5 pmol) was incubated in solution B with DnaJ, DnaK and GrpE. Then, the sample was centrifuged and supernatant was analyzed and the fraction of ribosomes after re-suspension of the latter in the same volume of solution B. Shot spectra and determined the number and activity of rhodanese in a small aliquot. Then each sample was added Chaperonin and GroES and the measurements were repeated. With regard to the fraction of ribosomes, after adding Chaperonin/ES sample was again centrifuged in the air centrifuge and determine the number and specific enzymatic activity.

Dramicanin, then this incubation mixture was centrifuged. Analyzed only obtained supernatant after the second incubation without mentors or preceptors.1-abbreviations listed in table II.

Example 12

Sparsomycin blocks the release associated with ribosomes CPM-rhodanese

Antibiotic sparsomycin associated with the subunits of the ribosome 50S with Kdapproximately 0.1 M and inhibits the peptidyl transferase reaction. At micromolar concentrations sparsomycin did not affect the fluorescence of coumarin from ribosomes carrying CPM-rhodanese. First sparsomycin and then mentors were added separately, as described in table III. Table VI shows that none of the mentors did not provide any informative steps on the measured fluorescence parameters. Adding mentors in the form of combinations (not shown) or separately after sparsomycin there were only very small changes in anisotropy compared with the data presented in table IIIA, which suggests that a large part of the polypeptides CPM - rhodanese remains associated with ribosomes. This was checked by centrifugation of the sample after performing a measurement of fluorescence. Less than 10% of the molecules regimentation inactive (data not shown). Thus, sparsomycin prevents the release rhodanese using DnaK or GroES from the ribosomes. Because sparsomycin inhibits the peptidyl transferase reaction, this invention demonstrates that CPM-rhodanese held on ribosomes in the form peptidyl-tRNA, and that the release with the help of DnaK and/or GroES involves the peptidyl transferase centre in the reaction, which hydrolyses the bond between the tRNA and the polypeptide.

Experimental conditions were the same as in table III, except that before adding each of the mentors individually added 4 μm sparsomycin. None of the mentors did not cause more than a few percent release CPM-rhodanese in the supernatant.

Example 13

The availability of the N-terminal coumarin for nitromethane

Quenching of fluorescence of peptides CPM-rhodanese nitromethane was evaluated as a measure of availability of this probe in different situations. Nitromethane is a deionized water-soluble small molecule, which effectively reduces the fluorescence of the coumarin in the solution when confronted with a probe. Curves stern-Volmer shown in Fig. 2. CPM-Met-f, free in solution and associated with ribelle available while it is largely protected from nitromethane, when CPM-Met-f associated with ribosomes. CPM at N-end of the enzymatic active rhodanese in solution was also relatively affordable. This is consistent with evidence of anisotropy for the enzymatic active rhodanese (table II) and crystal structure Rodney, in which the N-terminal segment is open. Interestingly, the damping was significantly lower for Rodney released from ribosomes DnaK or puromycin. This material was enzymatically inactive, as shown in tables III and V. the Values obtained in both cases were similar but lower than those obtained for free, enzymatically active rhodanese. Thus, this invention demonstrates that the N-end of this released material was less open than the native rhodanese and that this protein was not placed in its natural conformation. Even lower damping in the case associated with ribosomes rhodanese may be due partly shielded by ribosomes, especially the growing of short peptides, as well as incomplete laying full-length polypeptides.

Example 14

The intermediate state, detected IgG against coumarin

Polyclonal aerni Rodney, associated with ribosomes, in comparison with enzymatic active rodanthi found in free form in solution. Fig. 3A and 3B show that this probe was available for antibodies against coumarin as associated with ribosomes and free CPM-rhodanese. For the latter, the enzymatic active rhodanese was released from ribosomes by incubation with all the mentor, then the ribosomes were removed from the reaction mixture by centrifugation, as described in connection with table II. Antibodies cause an increase in anisotropy and quantum yield, which was associated with a bathochromic shift in fluorescence from both free and associated with ribosomes CPM-rhodanese. In the presence of IgG against coumarin maximum emission, approximately 456 nm was the same in both cases. The change in the quantum yield and the maximum emission, presumably reflects an increase in the hydrophobicity of the environment of the probe caused by the binding of immunoglobulin. The increase in the quantum yield caused by antibodies was lower in the case associated with ribosomes rhodanese (Fig. 3A) than in the case of enzymatic active CPM-rhodanese found in free form in solution (Fig. 3B). This difference in the quantum yield, possibly due to partial Ukranian ribosomes by polyalanine, and with the growing protein shell MS2. Similar to previous results with CPM-SAcAla-tRNA, CPM-SAcMet-f related site P ribosomes, apparently, completely shielded from IgG, since there is no change in any of the three options at incubation with IgG against CPM (data not shown).

To demonstrate mentors on associated with ribosomes growing CPM-rhodanese first sparsomycin, then DnaJ or DnaK, separately or together, were added to the reaction mixture containing the ribosome, such as those data are displayed in Fig. 3A. In the presence of sparsomycin of ribosomes were released very little Rodney, as discussed in connection with table VI. After incubation were added IgG against coumarin. The spectra of emission from the resulting mixtures are shown in Fig. 4. For comparison, the emission spectrum is given samples incubated only with sparsomycin (without adding mentors or IgG against coumarin). Chaperonin and GroES, added separately or together, did not give any spectral changes similar to the changes observed with DnaJ without DnaK or with DnaK in the presence of IgG.

Spectra for samples containing DnaK or DnaJ or both mentor, can be compared directly with the spectra of Fig. 3A, obtained for samples SOS IgG had little effect on the obtained emission spectrum, while clearly bimodal curve was observed for samples incubated with DnaJ. This effect was more pronounced for samples containing both DnaK and DnaJ. In this case, the distinct peaks visible in the spectrum at 435 nm and 472 nm. The latter is very close to the maximum for the free and bound with ribosomes rhodanese in the absence of IgG as shown in Fig. 3A and 3B.

Two peaks of the emission spectrum indicate that in the presence of IgG observed two types of flourescence in samples incubated with DnaJ or Dna and DnaK. This is because antibodies against coumarin able to interact only with a part of the CPM-rhodanese because DnaJ was associated with the N-end of some of the growing peptide Rodney. Hartl al. presented evidence suggesting that DnaJ may chemically be mixed with associated with ribosomes growing polypeptide. However, some evidence suggests that DnaJ is not associated with the N-end associated with ribosomes rhodanese (--), at least it detektirano does not affect the N-terminal coumarin. First, DnaJ had no visible action on the quantum yield when it is a separate Appendix, as well as anisotropy or emission spectrum, as shown in table III. Secondly, DnaJ apparently not the Zia quantum yield and anisotropy compared with the sample without DnaJ. Such a decrease was not observed (see the inset of Fig. 4 with the insert of Fig. 3A). What IgG is associated with coumarin in both types of fluorescence (maxima of approximately 440 nm and 470 nm, Fig. 4) is proved by similar changes in the intensity of fluorescence caused by IgG both regions of the emission spectrum. Thus, Fig. 4 shows that DnaJ causes a change in the condition or the growing peptide, or ribosomes, which affects the growing peptide in such a way that changes the environment of the probe, when it is associated with IgG. DnaK apparently facilitates this change caused DnaJ.

Example 15

Another aspect of the present invention the yeast TATA-block binding protein synthesized in the S30 extract of E. coli by coupled transcription/translation of plasmids containing the coding sequence under the control of the T7 promoter. In this case, the incubation mixture consisted of T7 RNA polymerase. The initiator tRNA was CPM-SAc[35S]Met-f.

10 μl of the reaction mixture synthesized approximately 125 ng N (coumarin) yeast protein that binds TATA-block. The reaction mixture was increased to 62 times. After incubation 600 ál reaction mixture, containing about 7.5 μg labeled indianinterracial acid (EDTA), 5 mm-mercaptoethanol and 20% glycerol (buffer A). Approximately 0,75 µg yeast TATA-block binding protein did not adsorbiroval on this matrix, approximately 1.5 µg N(coumarin)-binding protein TATA-block was suirable 200 mm KCl in the same buffer solution. Optional 3.0 µg coumarin labeled protein was suirable from the column 350 mm KCl. This material was applied to a heparin-Separato column (volume 1 ml), equilibrated in buffer A. Approximately half of radioactively labeled protein was adsorbiroval on the column and lirowaus by increasing the salt concentration to 0.7 M KCl in buffer A. Suirvey protein was used for studies using fluorescence binding of yeast TATA-block binding proteins with specific DNA sequences. As control was used mutated DNA sequence. Changes in the quantum yield of fluorescence of the coumarin and the anisotropy of the fluorescence indicate that synthesized in vitro protein purified as described above, specifically interacts with DNA containing the TATA-box.

With the application described here, a cell-free system broadcast protein synthesis takes place at a relatively high linear speed approximately in tecent ribosomes during the period of linear synthesis, but the disadvantage of this system is associated with the accumulation of apparently full-length polypeptides rhodanese in the form peptidyl-tRNA in the peptidyl transferase center of the ribosome. The existence of accumulated growing polypeptide in the form peptidyl-tRNA in the a site P ribosomes evidenced by their reactivity with puromycin and effect sparsomycin to block subsequent termination and release of protein. In the application here, the term "termination" refers only to those reactions that are directly related to hydrolysis of its ester link between the growing polypeptide and the tRNA, whereas the term "release" refers here to the separation of the protein from the ribosome. Accumulated associated with ribosome-nascent rhodanese was enzymatically inactive, but were released and turned into a fully active enzyme only by the incubation of ribosomes simultaneously with five mentors, DnaK, DnaJ, GrpE, Chaperonin and GroES. He was detected incorporating amino acids into the growing protein during this incubation under applied conditions. Rhodanese with reduced enzymatic activity was obtained by incubation of ribosomes first DnaJ, DnaK and GrpE, followed by a second incubation of these ribosomes with Chaperonin and GrpES. This, apparently, establishes the procedure for this is given or incubation only with DnaK or GroES, was enzymatically inactive. This is released from the ribosome protein activated by incubation with any combination of mentors. This is somewhat surprising, since Chaperonin. and GroES effectively increased re-laying in enzymatically active protein rhodanese, which was are denatured by urea or guanidine x HCl. The question mediashout whether Chaperonin and GroES or not mediashout laying protein on the ribosome or they act only on the released protein before it undergoes secondary reactions, which prevent the action of the Chaperonin and GroES, requires additional research.

Changes in fluorescence of the coumarin at N-end growing Rodney reflect dependent mentors reactions that take place during activation, termination and release of the enzyme from the ribosome. The fluorescence of the coumarin derivatives are very sensitive to the local environment of the fluorophore, so that changes in the position of the probe in the ribosome, laying the growing peptide to which it is linked or linking mentor near the probe, apparently, are reflected by changes in the quantum yield of fluorescence, emission maxima and anisotropy. Differences in these parameters for the fully enzymatic active CPM-rhodanese, the cat in the conformation of the protein. For example, you can compare the relative quantum yield of the enzymatic active soluble CPM-rhodanese (table II) with the release of enzymatically inactive soluble rhodanese, which was released from the ribosomes by incubation with DnaK or GroES (table III). It is not clear why these are clearly unrelated proteins with ATP enhances the release rhodanese. Their activity in this respect resembles the activity releasing factor 3, RF-3, which has a GTP-asnow activity.

What rhodanese exists in different conformations, is confirmed by Fig. 2, which illustrates the differences in the damping of the nitromethane fluorescence protein in different States. These differences probably reflect differences in the shielding of the probe by the protein in different conformational States. However, the striking feature is that none of the mentors, separately or in combination, did not cause pronounced changes in fluorescence, comparable to the change observed with IgG against coumarin (Fig. 3A and 3B). This apparently indicates that none of these mentors do not directly interact with the N-end CPM-rhodanese. Fig. 4 shows that DnaJ enhances the reaction, which affects associated with ribosomes exist in two different States, which may vary antibodies against coumarin. The component with the maximum emission near 440 nm is formed one DnaJ, but increases in the presence of DnaK, suggesting that the latter facilitates the reaction, the amplified DnaJ. It is possible that these results reflect the existence of growing chains of different length, for example, one kind or class which interacts with DnaJ or DnaK or both, whereas other species, presumably a shorter form, may not respond to these mentors. These two (hypothetical) class of different length molecules may represent different folding intermediates that exist in the lengthening of the growing peptide. A pronounced effect, visible in Fig. 4, was observed only in the presence of sparsomycin. Thus, the types of fluorescence with maxima near 440 and 470 nm represent the intermediate States generated by DnaJ and DnaK, which are caught by sparsomycin. Hence, they can be preceded by a codon-directed hydrolysis of the peptidyl-tRNA in the chain termination reactions. Applied mentors were highly purified and apparently free from releasing factors. A high proportion of releasing factors 1 and 2 was associated with ribosomes is by hydrolysis due to the peptide-tRNA, termination and release of the growing protein from the ribosome. The first reaction is blocked by sparsomycin and, as I believe, includes the peptidyl transferase reaction. A similar effect was observed with proteins produced in the endoplasmic reticulum. The relationship of particle recognition signal ribosome inhibits the elongation of the growing peptide up until the complex [particle recognition signal-increasing protein-ribosome] does not interact with the receptor or joining a protein on the membrane of the endoplasmic reticulum.

The question is, where in the ribosome happen reaction termination, installation and release of the protein. During the synthesis of polypeptides of the aminoacyl-tRNA and short peptides in the form of peptidyl-tRNA held rigidly in the hydrophobic site of the peptidyl transferase center, which is located at the base of the Central protuberance of the large subunit of the ribosome. tRNA associated with specific sites on the intermediate surfaces between the two subunits of ribosomes with the growing peptide in the form peptidyl-tRNA located in the peptidyl transferase center. However, electron microscopy shows that the domain of the output area in which the growing protein emerges from the ribosome, is located on the distal to the alanine peptide in the residues of alanine was used to estimate the distance from the peptidyl transferase center to the point, in which the N-terminal probe peptide were available for IgG or Fab fragments, approximately 60 residues or 90 (1,5 / amino acid residue in the helical conformation) for IgG and 40 residues or 60 for Fab. Fab fragment against coumarin derivatives can react with peptides shorter average length than the corresponding IgG, from which this fragment is received that suggests that its smaller size allows it to penetrate further into the ribosome. It was reported on the chemical crosslinking DnaJ with nascent peptide chloramphenicolchloramphenicol and luciferase Firefly, so short as 55 amino acids in length, on ribosomes wheat germ. The length of the peptide corresponds to the results for polyalanine above.

This invention shows that the amino-end of the short growing peptides screened within the domain of the ribosome as long as the peptides are not extended to the length at which their N-end will not be well removed from the site of the peptidyl transferase center. This domain may include a tunnel through the large subunit of the ribosome, as originally reported Jonath al. Eisenstein et al. (1994) did a review of data on the structure of ribosomes, indicating that the large subunits of the ribosome), (1993) presented data indicating that the signal sequence of a growing eukaryotic membrane proteins move through this tunnel during their synthesis. This invention assumes that the cavity inside the large subunit of the ribosome provides shelter, which may be at least part of the installation process, the environment in which the growing peptide partially shielded from proteases and other destructive processes that take place in the cytoplasm of the cell.

All patents and publications mentioned in this application, determine the levels of experts in this field, which converts this invention. These patents and publications is incorporated herein by reference, the same as if each individual publication was specifically and individually indicated as included in the reference.

The person skilled in the art will understand that the invention is well adapted to achieve the above objectives and obtain the above results and benefits, as well as other inherent advantages. Presents examples along with the methods, procedures, treatments, molecules, and specific compounds described herein are prepotencia. Changes therein and other uses that can meet professionals in the field, covered by the essence and scope of the invention defined by the scope of the claims.

1. The method of synthesizing a fluorescently-labeled protein in prokaryotic cell-free system for protein synthesis, which includes the following stages: (a) incubating the sample of ribosomes obtained from cell-free extract with plasmid DNA containing the coding sequence for the protein of interest, and the sample incubated in the medium for coupled transcription/translation together with the aminoacyl-tRNA with a fluorescent label, (b) partial purification of the indicated fluorescently-labeled protein separation of newly synthesized fluorescently-labeled protein from other fluorescent components in the sample, (b) measuring the amount of synthesized protein (g) determination of the fluorescence of the newly synthesized protein, (d) determining the biological activity of the newly synthesized protein.

2. The method according to p. 1, wherein the fluorescent label is on the N end of a specified protein.

3. The method according to p. 1, wherein said aminoacyl-tRNA is ecovalence connected with amino acid, the initiating aminoacyl-tRNA.

5. The method according to p. 1, characterized in that environment for the transcription/translation contains RNA polymerase, nucleosidases, system generation, rifampicin and amino acids in buffer containing saline.

6. The method according to p. 1, characterized in that the plasmid DNA is linear.

7. The method according to p. 1, characterized in that the incubation stage (a) is carried out for approximately 30 minutes at approximately 37oC.

8. The method according to p. 1, characterized in that the fluorescently-labeled protein partially purified by the method selected from the group consisting of centrifugation, ion exchange chromatography and gelfiltration.

9. The method according to p. 1, characterized in that the prokaryotic cell-free system for protein synthesis is a cell-free system for protein synthesis in E. coli.

 

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Thrombopoietin // 2245365

FIELD: medicine, molecular biology, polypeptides.

SUBSTANCE: invention describes homogenous polypeptide ligand mpI representing polypeptide fragment of the formula: X-hTPO-Y wherein hTPO has amino acid sequence of human fragments TPO (hML); X means a amino-terminal amino-group or amino acid(s) residue(s); Y means carboxy-terminal carboxy-group or amino acid(s) residue(s), or chimeric polypeptide, or polypeptide fragment comprising N-terminal residues of amino acid sequence hML. Also, invention relates to nucleic acid encoding polypeptide and expressing vector comprising nucleic acid. Invention describes methods for preparing the polypeptide using cell-host transformed with vector, and antibodies raised against to polypeptide. Invention describes methods and agents using active agents of this invention. The polypeptide ligand mpI effects on replication, differentiation or maturation of blood cells being especially on megacaryocytes and progenitor megacaryocyte cells that allows using polypeptides for treatment of thrombocytopenia.

EFFECT: valuable medicinal properties of polypeptide.

21 cl, 92 dwg, 14 tbl, 24 ex

FIELD: biotechnology, molecular biology.

SUBSTANCE: method involves transfection of cells HKB with vector pCIS25DTR comprising a selective marker and a sequence encoding protein eliciting procoagulating activity of factor VIII. Cells are selected using the selecting agent and clones with high level for expressing protein eliciting procoagulating activity of factor VIII are isolated. Invention provides preparing the protein eliciting activity of factor VIII with high yield, and strain of cells HKB with improved production under protein-free conditions also. Invention can be used for preparing the protein eliciting activity of factor VIII in industrial scale.

EFFECT: improved preparing and isolating methods.

8 cl,, 6 dwg, 1 tbl, 5 ex

FIELD: biotechnology.

SUBSTANCE: protein substances are produced from cultivation medium unlike prior art where they are produced by reprocessing of plant tissue. Application of moss protonema as plant tissue makes it possible to isolate therefrom heterologous proteins in active form without degradation of productive tissues and cells.

EFFECT: improved method for production of protein substances.

6 cl

FIELD: organic chemistry, peptides, medicine, pharmacy.

SUBSTANCE: invention relates to peptide derivatives named as memnopeptides that are used as an active component for manufacturing a medicinal preparation used in treatment of bacterial infection. Invention proposes compound of the formula (I): wherein radicals R1, R2, R3, R4, R5, R6, R7, R8 and (A)n have corresponding values, or its salt. Compounds of the formula (I) are prepared by culturing microorganism Memnoniella echinata FH 2272, DSM 13195 under suitable conditions in the nutrient medium containing at least one source of carbon atoms and at least one source of nitrogen atoms and the process is carrying out until the accumulation of at least one compound of the formula (I) in the nutrient medium followed by isolation of indicated compound. The attained technical result involves the development of a pharmaceutical composition eliciting an antibacterial activity. The development of the preparation provides expanding assortment of agents used in treatment of diseases said above.

EFFECT: improved preparing method, valuable medicinal properties of compounds.

10 cl, 2 tbl, 7 ex

FIELD: biotechnology.

SUBSTANCE: heterologous protein is obtained by cultivation of strain Corynebacterium glutamicus AJ 12036, which does not produce cell surface protein and contains gene expressing construct wherein nucleic acid sequence encoding signal peptide of cell surface protein of Corynebacterium glutamicus or C. ammoniagenes is bound in direct direction to promoter sequence, and nucleic acid sequence encoding heterologous protein is bound in direct direction to abovementioned nucleic acid sequence encoding signal peptide. Further heterologous protein secreted from cells is isolated.

EFFECT: high effective method for heterologous protein production.

6 cl, 8 tbl, 10 ex

FIELD: biotechnology, medicine, oncology, peptides.

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EFFECT: valuable medicinal properties of peptides.

2 dwg, 2 ex

FIELD: molecular biology, genetic engineering, polypeptides, medicine.

SUBSTANCE: in using the double-hybrid yeast system DNA sequences encoding polypeptides (55.1 and 55.3) have been found that elicit ability for binding with intracellular domain p-55 (p-55IC) of TNF-receptor. It has been established that these polypeptides represent fragments of amino acid sequences p-55IC, respectively, from 338 to 426 and from 277 to 426 residues. As result of insertion of DNA fragments with a sequence encoding polypeptide 55.1 or 55.3 into the structure of expressing vector and transformation suitable host-cells by this vector recombinant form of indicated polypeptides have been prepared. Using this invention provides the possibility for modulating the function of intact p-55 of TNF-receptor. Invention can be used in medicine in treatment of diseases associated with transfer of TNF-signal.

EFFECT: improved preparing method and valuable properties of polypeptide.

9 cl, 17 dwg, 3 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a method for preparing foodstuff containing hypotensive peptides and it using as an anti-hypertensive agent that can be used as a foodstuff. Method involves stages for fermentation of casein-containing fermenting material with lactobacillus microorganism, nanofiltration of the prepared peptide-containing fermentation product and isolation of the product. Prepared product is used as an anti-hypertensive agent and as a foodstuff also. Invention provides preparing a foodstuff with the high content of hypotensive peptides enriched by bivalent ions.

EFFECT: improved preparing method of foodstuff.

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FIELD: chemistry of peptides, microbiology, biotechnology.

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EFFECT: improved preparing method of dipeptide.

3 cl, 3 tbl, 1 ex

FIELD: gene engineering, in particular preparation based on PDGF-BB, useful in therapy, veterinary, diagnosis, tissue culturing.

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EFFECT: two-dimensional PDGF-BB form with high purity, specific activity and expression level in yeast.

5 cl, 11 dwg, 5 ex

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