EP1727897A1 - METHOD FOR PRODUCING RECOMBINANT RNase A - Google Patents

Abstract

The invention relates to a method for producing recombinant RNase A in E.coli. Said method is characterised in that it uses a DNA sequence, which codes for an RNase A of bovine origin and which is adapted to the codon usage in E.coli. The invention relates to nucleic acid molecules, which contain a nucleic acid sequence that has been adapted to the codon usage in E.coli and to recombinant nucleic acid molecules, which contain an nucleic acid molecule of this type and permit the expression of the recombinant RNase A in E.coli.

Description

 Process for the production of recombinant RNase A

The present invention relates to a method for producing recombinant RNase A in E. coli, which is characterized in that a DNA sequence is used which codes for an RNase A of bovine origin and which has been adapted to the codon use in E. coli. Furthermore, the invention relates to nucleic acid molecules which contain a nucleic acid sequence which has been adapted to the codon use in E. coli and to recombinant nucleic acid molecules which contain one of these nucleic acid molecules and which allow the expression of the recombinant RNase A in E. coli.

RNase A is an endoribonuclease that hydrolyzes RNA strands on internal phosphodiester bridges. It is specific for single-stranded RNA and cleaves 3 'bonds of pyrimidines. Therefore, form after the split

RNase A pyrimidine 3 'phosphates and oligonucleotides with terminal pyrimidine 3' phosphates. RNase A consists of a chain of 124 amino acids intramolecularly cross-linked by four disulfide bridges. RNase A is enzymatically active even in the absence of co-factors and divalent cations. It is inhibited by heavy metal ions as well as by DNA in a competitive way.

RNase A is used in various molecular biological techniques. When isolating either plasmid DNA from bacterial cells or genomic DNA from eukaryotic cells, for example, RNA is also purified in addition to DNA, which in large quantities leads to an increased viscosity of the sample and a reduction in the yield. Therefore, the RNA must be degraded by adding RNase A to increase the quality and quantity of the sample. The same applies to the preparation of recombinant proteins. RNase A is also used for the detection of single-base mutations in RNA or DNA. In this case, RNase A cleaves on mismatches, for example in RNA-RNA heteroduplexes, which were formed between a reference wild-type RNA and a possibly mutated RNA. The size of the split strand can then be estimated by gel electrophoresis.

Finally, RNase A is also used in RNase protection assays with which the expression of different genes can be examined simultaneously. This method is based on the hybridization of sample RNAs to complementary, radioactively labeled RNA probes (ribo samples) and the subsequent digestion of non-hybridized sequences with one or more single-stranded ribonucleases. After the digestion has taken place, the ribonucleases are inactivated and the protected fragments of the radioactively labeled RNA are analyzed by polyacrylamide gel electrophoresis and autoradiography.

The variety of molecular biological applications for RNase A requires the isolation of large amounts of the enzyme in high purity. The classic way of producing RNase A involves isolation from the bovine pancreas. However, the BSE problem of recent years has resulted in animal raw materials, especially those derived from cattle, no longer being accepted by the authorities in pharmaceutical production for reasons of biological safety. For this reason, the use of RNases has been completely dispensed with in recent years and the RNA in many biotechnological-pharmaceutical processes has instead been separated off using alternative, usually very expensive, processes such as chromatography. Therefore, there is a need for a method that enables the production of large amounts of RNase A without the use of animal material. This can be achieved primarily by the recombinant production of RNase A.

However, recombinant production of RNase A is complicated by four factors: (1) RNase A is unstable when expressed alone in E. coli; (2) In order to reconstitute RNase A into an active protein, four disulfide bridges must be correctly formed; (3) RNase A expression within a cell may be cytotoxic, and (4) RNase A may break down its own transcript, causing expression performance to decrease accordingly.

In the past, various possibilities for recombinant expression of RNase A have been tried, which should overcome these difficulties, but which all led to a rather low RNase A yield.

In one approach, RNase A was expressed under the control of a heat-inducible promoter. This resulted in the formation of inclusion bodies and a yield of about 2 mg / l (McGeehan and Brenner (1989) FEBS Letters 247 (1): 55-56).

The expression of a fusion protein of RNase A with a gene 10 protein from bacteriophage T7 under the control of an IPTG-inducible promoter also led to the formation of inclusion bodies. After enzymatic cleavage of the fusion protein with the protease factor Xa and purification, a yield of 4-8 mg / l protein was achieved (Laity et al. (1993) Proc. Natl. Acad. Sci. USA 90: 615-619). A fusion protein from β-galactosidase and RNase A was also expressed in E. coli under the control of the IPTG-inducible β-galactosidase promoter. With this strategy, a yield of 0.2 mg / l was achieved after purification (Nambiar et al. (1987) Eur. J. Biochem. 163: 67-71).

RNase A was also expressed together with a signal peptide which brings about the efficient translocation of RNase into the periplasm under the control of an IPTG-inducible promoter. RNase A was released from the periplasm by spheroplast / osmotic Schoc-lc and purified. With this strategy, a yield of 0.1 nxg / l was achieved (Tarragona-Fiol et al. (1992) Gene 118: 239-245).

Finally, a combination of heat-inducible promoter and a signal peptide that controls the transport of RNase A into the periplasm was also tested in E. coli cells. The periplasmic proteins were also examined here

Spheroid plastic / osmotic Schoc-k released and cleaned. With this method, a yield of 45-5O mg / 1 could be achieved (Okorokov et al. (1995) Protein Expression and Purification 6: 472-480).

Host cells other than E. coli, e.g. Bacillus subtilis and Pichia pastoris were used to express RNase A. With these host cells, yields of the order of 1-5 mg / l could also be achieved (Vasantha and Filpula (1989) Oene 76: 53-60; Chatani et al. (2000) Biosci. Biotechnol. Biochem. 64 (11) : 2437-2444).

Thus, despite multiple attempts to optimize recombinant RNase A expression, there is a need for a method that can produce enables recombinant RNase A in E. coli with a higher yield than in the prior art.

The object of the invention is therefore to provide a method by means of which recombinant RNase A can be produced in large quantities in E.co cells.

These and other objects are achieved according to the invention by the features of the main claim.

Advantageous refinements are defined in the subclaims.

According to the invention, a method is provided for the production of recombinant RNase A in E. coli, which is characterized in that a DNA sequence is used which codes for an RNase A of bovine origin and which has been adapted to the codon use in E. coli.

The genetic code is redundant because 20 amino acids from 61 triplet codons are specified. Therefore, most of the 20 proteinogenic amino acids are encoded by several base triplets (codons). However, the synonymous codons that specify a single amino acid are not used with the same frequency in a particular organism, but there are preferred codons that are used frequently and codons that are used less frequently. These differences in codon usage are attributed to selective evolutionary pressures and, above all, the efficiency of translation. One reason for the lower translation efficiency of rarely occurring codons could be that the corresponding aminoacyl tRNA pools are exhausted and are therefore no longer available for protein synthesis. In addition, different organisms prefer different codons. For example, the expression of a recombinant DNA that originates from a mammalian cell in E. co / t cells is often only suboptimal. Therefore, replacing rarely used codons for commonly used codons can increase expression in some cases.

For many organisms, of which the DNA sequence of a larger number of genes is known, there are tables from which the frequency of the use of certain codons in the respective organism can be seen. With the help of these tables, protein sequences can be translated back with relatively great accuracy into a DNA sequence which contains the codons preferred in the respective organism for the different amino acids of the protein. Tables for codon usage can include can be found at the following Internet addresses: http: //www.kazusa.or.ip/Kodon/E.html; http://www.hgmp.mrc.ac.uk/Software/EIVlBOSS/Apps/cai.html; http://www.hgmp.mrc.ac.uk/Software/EIV-BOSS/Apps/chips.html; or http: // www. Entelechon. com / eng / cutanalysis .html. Programs are also available for the reverse translation of a protein sequence, for example the protein sequence of RNase A, into a degenerate DNA sequence, such as at http://www.entelechon.com/eng/backtranslation.html; or http: //wΛvw.hgmp.mrc.ac.uk/Software.EMBOSS/Apps/backtranseq.html.

In the method according to the invention, the DNA sequence used to express the recombinant RNase A was adapted to the codon use of the E.co/t strain Kl 2.

The sequences can be adapted to the codon use in a particular organism with the aid of various criteria. On the one hand the codon most frequently occurring in the selected organism can always be used for a certain amino acid, - on the other hand the natural frequency of the different codons in the selected organism can also be taken into account, so that all codons for a certain amino acid according to their natural frequency in the genome of the selected organism in the optimized sequence. The choice of the position at which base triplet is used can be random. Both strategies for optimizing the DNA sequence with regard to the use of codons have proven equally suitable for the method according to the invention.

The DNA sequence coding for the bovine RNase A is in at least 30 positions, preferably in at least 40 positions, particularly preferably in at least 50 positions and most preferably in at least 60 positions with respect to the codon use in the E. coli sta m Kl - 2 optimized.

Most preferably, the optimized DNA sequences are those in SEQ ID No. 1 or SEQ ID No. 2 DNA sequences shown or DNA sequences that correspond to those in SEQ ID No. 1 or SEQ ID No. 2 DNA sequences shown are at least 90, preferably at least 92 or 94%, particularly preferably at least 96 or 98% and most ^" preferably at least 99% identical over the entire coding sequence.

Sequence identity is determined by a number of programs based on different algorithms. The algorithms from Needleman and Wunsch or Smith and Waterman deliver particularly reliable results. The program PileUp (Feng and Doolittle (1987) J. Mol. Evolution 25: 351-360; Higgins et al. (1989) CABIOS 5: 151-153) or the Gap and Best Fit programs (Needleman and Wunsch (1970) J. Mol. Biol. 48: 443-453 and Smith and Waterman (1981) Adv. Appl. Math. 2: 482-489) used, which are included in the GCG software package (Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA).

The sequence identity values given above in percent were determined with the GAP program over the entire sequence range with the following settings: Gap Weight: 50, Length Weight: 3, Average Match: 10000 and Average Mismatch: 0.000.

Unless otherwise stated, these settings were used as standard settings for sequence comparisons.

Without wishing to be bound by a hypothesis, it is assumed that the codon-optimized DNA sequences enable more efficient translation and the mRNAs formed therefrom may have a higher half-life in the cell and are therefore more frequently available for translation.

An "RNase A of bovine origin" has essentially the same

Amino acid sequence like the native protein occurring in bovine cells, but is not encoded by the DNA occurring in the bovine cells.

In a preferred embodiment, the RNase A is expressed in fusion with a signal peptide that controls the transport into the periplasmic space. Localization in the periplasmic space prevents possible cytotoxic effects that could arise from the expression of RNase A. Examples of such signal peptides include stll and phoA (Denefle et al. (1989) Gene 85: 499-510), ompF and LamB (Hoffman and Wright (1985) Proc. Natl. Aca-d. Sei. USA 82: 5107-5111), pelB (Lei et al. (1987) J. Bacteriol. 169 (9) : 4379-4383), OmpT (Johnson et al. (1996) Protein Expression Purif. 7: 104-113), Beta-lactamase (Kadonaga et al. (1984) J. Biol. Chem. 259: 2149-2154), Enterotoxins Lr-A, LT-B (Morioka-Fujimoto et al. (1991) J. Biol. Chem. 266: 1728-1732) and Protein A from S. aureus (Abrahmsen et al. (1986) Nucleic Acids Res. 14 : 7487-7500).

Various non-natural, synthetic signal sequences which enable the secretion of certain proteins are also well known to the person skilled in the art.

The phoA signal peptide is preferably used.

The expression of RNase A is preferably under the control of an incusible promoter. A heat-inducible promoter is particularly preferably used, in which the expression of the gene under its control is induced by increasing the cultivation temperature to 42 ° C.

The invention also relates to a recombinant nucleic acid molecule comprising the following constituents in 5 '-3' order: - a promoter active in E. coli, - optionally a sequence coding for a signal peptide, - one for the codon use in E. coli-matched DNA sequence encoding an RNase A of bovine origin.

The promoter is preferably an inducible proinotor, particularly preferably a heat-inducible promoter. The signal peptide is preferably a signal sequence which is responsible for the transport of the protein in controls the periplasmic space, and particularly preferably around the phoA signal peptide.

The methods for producing a nucleic acid molecule which comprises the components listed above belong to the molecular biological

Standard methods and can be used in literature such as Sambrook and Russell (2001) Molecular Cloning - A laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA.

The induction of the promoter takes place preferably in the middle to the end of the exponential growth phase. At this point in time, the cells have reached a bio-moist mass of approximately 35 to 50 g / l culture medium. Protein expression is induced for at least 14, usually for 14 to 20 hours, preferably for at least 16, usually for 16 to 18 hours, and most preferably for about 17 hours.

Various E.co/z ^' strains are suitable as host cells for the expression of the recombinant RNase A, including BL21, BNN93, MM294, ATCC 23226 and ATCC 23851.

Methods are known to the person skilled in the art with which he can introduce the DNA for the recombinant expression of RNase A into the host cells. These methods include chemical methods as well as physical methods such as electroporation. Suitable culture media and culture conditions for the E. eo / z cells are also known to the person skilled in the art. These can also be found in the literature, such as Sambrook and Russell (2001) Molecular Cloning - A laboratory manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA. The E. co t cell culture in a suitable culture medium contains at least 0.2 g RNase A per liter of culture medium, preferably at least 0.5 g / 1, particularly preferably at least 1 g / 1, during the harvest after cultivation and, if appropriate, induction most preferably about 1.2 g RNase A per liter of culture medium.

In a preferred embodiment, RNase A forms inclusion bodies in the host cells. These inclusion bodies are insoluble intracellular aggregates of the expressed protein. They can be isolated by centrifugation at low speed and usually consist of almost pure ones

Deposits of denatured forms of the recombinantly expressed protein. In the case of RNase A, the formation of the inclusion bodies may contribute to the high yield, since the product is in an inactive form and therefore cannot show any cytotoxic effects.

To release the inclusion bodies, the host cell must be lysed. Cell lysis can be accomplished e.g. by mechanical shear stress, enzymatic digestion, e.g. with lysozyme, ultrasound treatment, homogenization, glass ball vortexing, treatment with detergents or organic solvents, by freezing and thawing or by treatment with a

Denaturant (Bollag et al. (1996) Protein Methods, 415 pages, Wiley-Liss, NY, NY). Optionally, the cells can be lysed in the presence of a denaturing agent or a disulfide reducing agent. Insoluble or aggregated material can be separated from soluble proteins by various methods, such as centrifugation, filtration (including ultrafiltration) or precipitation. In the next step, the insoluble or aggregated material must be made soluble or monomeric by treating it with a denaturing agent. Suitable denaturing agents include: urea, guanidine, arginine, sodium thiocyanate, pH extremes (dilute acids or bases), detergents (eg SDS, sarcosyl), salts (chlorides, nitrates, thiocyanates, trichloroacetates), chemical derivatization (sulfitolysis, reaction with citraconanhydride ), Solvents (2-amino-2-methyl-l-propanol or other alcohols, DMSO, DMF) or strong anion exchange resins such as Q-Sepharose. Suitable concentrations of urea are 1 to 8 M, preferably 5 to 8 M. Suitable concentrations of guanidine are 1 to 8 M, preferably 4 to 8 M. Guanidine is particularly preferably used in a concentration of 5 M.

The solubilization buffer particularly preferably additionally contains a redox mixture of an oxidizing agent and a reducing agent in order to promote the reduction of intra- and intermolecular disulfide bridges. Examples of suitable ones

Redox mixtures include cysteine oxygen, cysteine / cystine, cysteine / cystamine, cysteamine / cystamine and reduced glutathione / oxidized glutathione. Most preferably, the solubilization buffer contains reduced and oxidized glutathione. The reduced glutathione is present in the solubilization buffer in a concentration of 1 to 10 mM, preferably 2 to 5 mM. The concentration of the oxidized glutathione is 1/10 to 1/1, preferably 1/10, of the concentration of the reduced glutathione.

The pH of the solubilization mixture is preferably between pH 6 and pH 10, particularly preferably between 7.5 and 9.5, most preferably around pH 9. After solubilizing the inclusion bodies, the protein must be folded back into its active form. To do this, the protein must adopt its native conformation and form its native disulfide bridges. Refolding is accomplished by reducing the concentration of the denaturant so that the protein can renaturate into its soluble, biologically active form. The concentration of the denaturant can be reduced by dialysis, dilution, gel filtration, precipitation of the protein or by immobilization on a resin, followed by washing with a buffer. The concentration of the denaturing agent is preferably reduced by dilution in a native buffer.

In order to reverse the native disulfide bridges of the protein, an oxidizing agent or a redox mixture of an oxidizing agent and a reducing agent is added which catalyze the disulfide exchange reaction. Suitable oxidizing agents include oxygen, cystine, oxidized glutathione, cystamine and dithioglycolic acid. Examples of suitable redox mixtures include cysteine / oxygen, cysteine / cystine, cysteine / cystamine, cysteamine / cystamine, reduced glutathione / oxidized glutathione, sodium sulfite / sodium tetrathionate, etc. If necessary, a reducing agent such as DTT or 2-mercaptoethanol can be added to the refolding mixture To request disulfide exchanges. Optionally, a metal ion such as copper can be added to the refolding mixture to promote oxidation of the protein. Suitable concentrations of metal ions in the refolding mixture are 1 μM to 1 mM.

The refolding mixture preferably contains reduced and oxidized glutathione. The reduced glutathione is present in a concentration of 1 to 10 mM, preferably 2 to 5 mM, in the refolding mixture. The concentration of the oxidized glutathione is 1/10 to 1/1, preferably 1/10, of the concentration of the reduced glutathione. The pH of the refolding mixture is preferably between pH 6 and pH 10, particularly preferably the pH is between 7.5 and 9.5 and most preferably the pH of the refolding mixture is approximately pH 9.

After the RNase A has been solubilized and refolded, it is preferably further purified by chromatographic steps. Chromatography is particularly preferably a cation exchange chromatography, in which the RNase A at a specific pH of the buffer, which should be at least 0.5 to 1.5 pH units below the pI value of the RNase A of 9.45 , binds to the matrix of the cation exchange column with its positive total charge, while most contaminating proteins do not bind and can be removed by washing.

Suitable cation exchange matrices include carboxymethyl (CM) cellulose, AG 50 W, Bio-Rex 70, carboxymethyl (CM) -Sephadex, sulfopropyl (SP) -Sephadex, carboxymethyl (CM) -Sepharose CL-6B and sulfonate (S) -Sepharose ,

The person skilled in the art can find suitable matrices and protocols for carrying out the cation exchange chromatography from the product information from providers such as Amersham Biosciences (http://www.amershambiosciences.com) or Bio-Rad (http://www.bio-rad.com).

Suitable buffers for cation exchange chromatography include maleate, malonate, citrate, lactate, acetate, phosphate, HEPES and bicin buffers. The

The concentration of the buffer is preferably between 20 mM and 50 mM. For the purification of the RNase A, the pH of the buffer should preferably not be higher than 8.0, preferably not higher than 7.0. 20 mM sodium acetate pH 5.0 or 50 mM Tris-HCl pH 6.8 is particularly preferably used for the cation exchange chromatography.

After washing, the RNase A can be eluted from the column by a change, in the case of cation exchange chromatography, an increase in the pH or an increase in the ionic strength.

Elution is preferably effected by increasing the ionic strength. If 20 mM sodium acetate pH 5.0 is used as a buffer, a mixture of 20 mM sodium acetate pH 5.0 and 350 mM NaCl is used for elution. If 50 mM Tris-HCl pH 6.8 is used as a buffer, Tris-HCl pH 6.8 is used for the elution in a concentration of 250 mM.

Further suitable conditions for cation exchange chromatography can be found in the relevant literature, such as the manual "Ion Exchange Chromatography Principles and Methods" from Amersham Biosciences, Freiburg, Germany.

The RNase A can then be further purified by additional chromatographic steps as well as filtration, precipitation and diafiltration.

After the purification, DNases purified with RNase A may have to be inactivated by heat-treating the product-containing chromatography fractions at 95 ° C in a water bath. The heat treatment is preferably carried out for 20 to 35 minutes, particularly preferably for about 20 minutes. Alternatively, the heat treatment can also be carried out at 80 ° C over a correspondingly extended period. After purification, the RNase A can be analyzed for its amount and its activity. The quantity of the purified RNase A can be analyzed qualitatively on the one hand by means of an SDS-PAGE analysis and subsequent Coomassie Brilliant Blue staining. A colorimetric assay such as the Bradford assay or a chemical reaction such as the Lowry or Biuret reaction can be used to quantify RNase A. A non-recombinant, commercially available bovine RNase A with a known combination can be used as the standard for the analyzes. In addition, the concentration of the protein solution can also be below the absorbance at 278 nm

Taking into account the molar extinction coefficient of RNase A can be calculated.

With the method according to the invention, a yield of RNase A of more than 100 mg / 1 culture medium, preferably of more than 200 mg / 1 culture medium, particularly preferably of more than 250 mg / 1 culture medium and most preferably of about 300 mg, is obtained after the purification / 1 culture medium reached.

With the method according to the invention, after the purification, a yield of RNase A of more than 3 mg / g of bio-moist mass, preferably of more than 4 mg / g of bio-moist mass, more preferably of more than 6 mg / g of bio-moist mass and most preferably of more than 8 mg / g bio-moist mass reached.

The activity of RNase A can be determined by digestion of defined RNA molecules. RNase A activity is expressed in Kunitz units. A Kunitz unit causes an absorbance decrease at 300 nm of 100% in one minute at a temperature of 25 ° C and a pH of 5.0 when total RNA from yeast is used as the substrate. To compare the activity of the RNase A purified according to the method of the invention with commercially available, non-recombinant RNase A preparations, the commercially available RNase A preparations can be used as standard.

The RNase A purified by the process according to the invention has an activity of at least 40 Kunitz units and preferably of at least 50 Kunitz units. This activity is comparable to the values given by the manufacturers for non-recombinant RNase A.

Alternatively, the activity test can also be carried out with other RNAs at different temperatures and over different time periods. In addition, it is of course possible to test the activity of RNase A in the context of the desired end use, e.g. in the form of plasmid isolation.

The purified RNase A can either be stored as a lyophilisate or in liquid form. Suitable buffers for liquid storage are e.g. Tris-HCl at pH 6.8 to 7.4, a phosphate buffer or sodium acetate. In addition, the buffers can contain other components such as glycerin, Triton X-100, sodium chloride or EDTA. If Tris-HCl is used as a buffer, it is used in a concentration of 10 mM to 250 mM. It is preferably used in a concentration of 250 mM. RNase A is present in the solution in a concentration of 1 to 100 mg / ml, preferably 100 mg / ml.

The RNase A produced by the method according to the invention is suitable for all applications in which RNase A is usually used, for example for the isolation of plasmid or genomic DNA and recombinant proteins, the detection of single-base mutations or the ribonuclease protection assay. The present invention is illustrated by the following examples, which are not to be understood as a limitation.

Examples:

1. Optimization of the DNA coding for RNase A

To optimize the use of codons, the protein sequence of the mature RNase A, which can be found under the accession number AAB35594 in the NCBI protein database (http://www.ncbi.nlm.nih.gov/entrez), was reverse-translated. This resulted in a degenerate DNA sequence, which was subsequently optimized for E.coli K12 cells with the help of publicly available codon usage tables (http://www.kazusa.or.Jp/Kodon/E.html). The DNA sequence for RNase A was adapted to the codon use in the E. co / t strain K12 by always using the codon most frequently used in these cells for a certain amino acid. The optimization took into account that no additional Ndel- or Sall restriction sites were generated, as these would have made later cloning into the expression vector more difficult. The cDNA sequence thus obtained, which was designated as mRAopt and is described in SEQ ID No. 1 is shown, was synthesized by Geneart (Regensburg, Germany).

The phoA signal peptide comes from the alkaline phosphatase from Lysobacter enzymogenes (Accession No. Q05205). The sequence of the putative signal peptide (29 amino acids) was determined using an Entelechon applet (http://www.entelechon.coin/) on their homepage, taking into account the Codon usage in E. coli Kl 2 reverse translated. Derived from this reversely translated nucleic acid sequence, two single-stranded DNA oligonucleotides (phoA for, phoA rev) were produced and linked to the coding cDNA mRAopt via recombinant PCR.

2. Cloning of the optimized DNA into an expression vector and transformation of the E. co // cells

In a so-called primary PCR reaction, the two were used for the

Oligonucleotides phoA for (SEQ ID No. 3) and phoA rev (SEQ ID No. 4) encoding signal peptide hybridized to a double strand and amplified under the following conditions:

Reaction approach:

10 μl phoA for (10 pmol / μl) 10 μl pho A rev (10 pmol / μl)

10 μl lOx PCR buffer with MgCl ₂ (15mM) (Expand High Fidelity PCR System, Röche, Mannheim, Germany)

4 μl dNTP mix (2 mM each) (Gibco Life Technologies, Eggenstein, Germany) 1 μl polymerase (3.5 units / μl) (Expand High Fidelity PCR System, Röche, Mannheim, Germany) 65 μl H ₂ O PCR conditions:

1st step: 1 min 95 ° C

2nd step: 1 min 95 ° C 30 sec 50 ° C 30 sec 72 ° C 25 cycles

3rd step: 4 min 72 ° C

4th step: 4 ° C

The reaction product of the primary PCR reaction was carried out together with the plasmid which contains the optimized DNA sequence (pmRAopt, supplied by Geneart) and a primer which carried out the amplification from the 3 'end of the

RNase A-opt cDNA enables (5 * GTC GAC TAT TAG ACG CTC GCA TC 3 '), used in a so-called recombinant PCR reaction with the following conditions:

Reaction:

10 μl pmRAopt (10 ng / μl)

10 μl RNase opt 3 'primer for amplification (10 pmol / μl) 10 μl primary PCR product 10 μl 1 Ox PCR buffer with MgCl ₂ (15 mM) (Expand High Fidelity PCR System, Röche, Mannheim, Germany) 8 μl dNTP mix (2 mM each) (Gibco Life Technologies, Eggenstein, Germany) 1 μl polymerase (3.5 units μl) (Expand High Fidelity PCR System, Röche, Mannheim, Germany)

PCR conditions:

1st step: 1 min 95 ° C

2nd step: 1 min 95 ° C 30 sec 55 ° C 45 sec 72 ° C 30 cycles

3rd step: 4 min 72 ° C

4th step: 4 ° C

In the PCR product obtained by this reaction, the DNA sequence for the signal peptide was at the 5 'end of the RNase A opt cDNA.

The PCR product was purified on an agarose gel and ligated into the vector pCR2.1-TOPO (Invitrogen, Karlsruhe, Germany). The correct sequence was verified by sequencing. The cloned PCR product was then cut out of the vector using an N- / Sα / I double digest and purified using an agarose gel. In parallel, the vector pHIP-pelB-R-ase-opt with NdeVSaU. digested, yielding the linearized vector pHIP with Ndel and S / I ends. This vector was also cleaned on an agarose gel. The NdellSall-digested pho A-RΝase A-opt fragment was ligated into the linearized vector and transformed into E. coli TOPIOF 'cells and amplified. The plasmid DNA was then isolated from this strain and used for the calcium chloride-mediated transformation of the E. co / z 'strain ATCC 23226 (cf. Sambrook and Russell, vide suprd).

The methods used here for cloning the optimized cDNA are well known to the person skilled in the art and can be found, for example, in Sambrook and Russell (2001), vide supra.

The map of the expression vector pHIP is shown in Fig. 1, the sequence of the expression vector is shown in SEQ ID No. 5 specified. This nector already contains the heat-inducible promoter sequence and a multiple cloning site into which corresponding cDΝAs can be cloned via the restriction sites of the enzymes Ndel and Sau.

Fermentation and induction of the bacteria

The bacteria were in complete medium (soy peptone (27 g / 1), yeast extract (14 g / 1), ΝaCl (5 g / 1), K ₂ HPO ₄ (6 g / 1), KH ₂ PO ₄ ( 3 g / 1), MgSO ₄ (0.5 g / 1), glycerin (30 g / 1)) cultured at 30 ° C. At certain time intervals, 1 ml of culture broth was transferred to a previously weighed Eppendorf reaction vessel and the cells were pelleted by centrifugation at 13000 xg for 3 minutes. The supernatant was removed completely and the vessel was weighed again. From the difference between the weight of the reaction vessel with cell pellet [mg] and the weight of the empty reaction vessel [mg] divided by the volume of the culture broth from which the cells were pelleted, the bio-moist mass can be determined [mg / ml or g / 1] determine. The determination was carried out as a double determination. After reaching a bio-moist mass of 35 to 50 g / 1, the cultivation temperature was raised to 42 ° C increased, which induced the expression of the recombinant protein. The induction phase lasted 17 hours.

4. Harvesting the bacteria and obtaining the native RNase A from the inclusion bodies

After the induction phase, the cells were harvested by centrifugation and resuspended in 20 mM sodium phosphate buffer pH 7.4, which was used in an amount of 3.75 ml / g of bio-moist mass. The cells were then physically disrupted through two passages in the high-pressure homogenizer (Niro Soavi, Lübeck, Germany) at a pressure of 800 ± 50 bar. The inclusion body fraction was harvested by centrifugation (11000 xg for 30 minutes at 4 ° C.), resuspended in 20 mM sodium phosphate buffer pH 7.4, the buffer being used in an amount of 5 g / 1 inclusion bodies, and again Centrifugation pelleted.

The inclusion body fraction was adjusted in a denaturing redox buffer (5 M guanidine HCl, 50 mM Tris, 1 mM EDTA, 50 mM NaCl, 2 mM glutathione (reduced), 0.2 mM glutathione (oxidized), pH 9) NaOH or HCl) solubilized.

Insoluble constituents of the inclusion body fraction were removed from the mixture by centrifugation or filtration. The optical density of the clarified solution was then determined at 280 nm (OD _280mn ).

The denatured proteins were refolded by dilution in a native dilution buffer (50 mM Tris, 1 mM EDTA, 50 mM NaCl, 2 mM Glutathione (reduced), 0.2 mM glutathione (oxidized), adjusted to pH 9 with NaOH or HCl). The volume of the dilution buffer was chosen such that an OD280 _mn of 5 resulted in the refolding batch after adding the solubilized inclusion body fraction. This batch was gently stirred for at least 15 hours (150 to 180 revolutions per minute).

5. Ion exchange chromatography

The refolding batch was clarified by filtration and then concentrated and buffered by ultrafiltration, so that the batch was in a buffer suitable for the subsequent chromatography (50 mM Tris-HCl pH 6.8). The batch obtained from the ultrafiltration was purified by chromatography using a cation exchanger (SP-Sepharose, company Amersham Pharmacia, Freiburg, Germany). Non-specific proteins were removed by washing with approximately three column volumes of 100 mM Tris-HCl pH 6.8. Elution was carried out by increasing the salt concentration to 250 mM Tris-HCl pH 6.8.

6. Heat inactivation and bottling of RNase A

The product-containing chromatography fractions were identified by measuring the absorbance at 280 nm in the chromatography system, combined and heat-treated in a water bath at 95 ° C. for 20 minutes. Precipitate was removed after the heat treatment by filtration or centrifugation. The sample was concentrated by ultrafiltration and the RNase A solution was adjusted to a final concentration of 10O mg / ml by adding 250 mM Tris-HCl pH 6.8. The solution was filtered and filled sterile. 7. Analysis of the purified RNase A

The recombinant, purified RNase A was qualitatively analyzed by SDS-PAGE analysis with subsequent Coomassie brilliant blue staining as described in Sambrook and Russell (2001), vide supra.

Figure 2 shows the analysis of protein expression at different times of cultivation or induction. The expression of the recombinant RNase A is detectable as early as 1.5 hours after induction and increases up to 18 hours after induction.

Figure 3 shows the SDS-PAGE analysis of RNase A after the individual purification steps. Only the RNase A is purified using the sequence of purification steps described above.

The concentration of the purified RNase A was determined colorimetrically according to Bradford, M.M. ((1976) Anal. Biochem. 72: 248-254).

To analyze the RNase activity, different amounts of the RNase A produced by the method according to the invention were mixed with 10 μl total yeast RNA (10 μg / μl in 100 mM sodium acetate pH 5.0) in a total volume of 20 μl for 5 minutes at room temperature (20 ° C) incubated. For comparison, a commercially available RNase A was analyzed in parallel. The treated samples were mixed with 4 μl loading buffer, electrophoresed on a 1% agarose gel in 1 × TAE and visualized by staining with ethidium bromide. There was no significant difference between the two RNase A preparations (see Fig. 4a). In addition, both the RNase A, which had been prepared by the process according to the invention, and two different commercially available RNase A preparations were used to purify two different plasmids. For this purpose, the corresponding cultures were incubated overnight in dYT medium with kanamycin (50 μg / ml) at 37 ° C. and 180 revolutions per minute. 1.5 ml of each culture were transferred to an Eppendorf reaction vessel per batch and pelleted in a table centrifuge for 3 min at 13,000 revolutions per minute. The pellets were resuspended in 200 μl buffer 1 (Qiagen, Hilden, Germany; 50 mM Tris-HCl (pH 8), 10 mM EDTA, 100 μg / ml RNase A), with 200 μl buffer 2 (Qiagen; 0.2 M NaOH, 1% (w / v) SDS) mixed and incubated for 3-5 min at room temperature. 200 μl of buffer 3 (from Qiagen; 3 M potassium acetate pH 5.5) were then added, the samples were mixed by inverting and centrifuged for 5 min in a table centrifuge at 13,000 revolutions per minute. The supernatant (500 μl) was transferred to a new reaction vessel. 10 μl of each batch were applied to a 1.2% agarose gel in 1 × TAE, separated electrophoretically and then stained with ethidium bromide. No difference with regard to the RNase A activity or the DNA conformation could be found in the batches with the RNase A according to the invention and the commercial RNase A preparations (see FIG. 4b).

pictures

1. Map of the pHIP expression vector

The components of the expression vector and the interfaces of the restriction enzymes are marked.

2. Investigation of the kinetics of the expression of the recombinant RNase A in the E. co / z cells used by SDS-PAGE analysis

1: Prestained marker (NEB Beverly, MA, USA), 2: 1h before induction, 3: 0.5h before induction, 4: Induction, 5: 0.5h after induction, 6: 1.5h after induction, 7: 2.5 h after induction, 8: 18 h after induction

SDS-PAGE analysis of RNase A after the various purification steps

1: Prestained marker (NEB, Beverly, MA, USA), 2: Cell harvest, 3: soluble proteins, 4: supernatant after washing the inclusion body fraction, 5: solubilized inclusion body fraction, 6: after refolding, 7: after diafiltration, 8: flow through chromatography (= unbound material), 9: eluate 1, 10: eluate 2, 11: eluate 3, 12: commercial, bovine RNase A 4. Activity tests of the purified RNase A a) digestion of RNA

1, 15: 1 kb size marker, 2-7; different amounts of commercial, bovine RNase A (from 1 μg to 0.025 μg), 8: control without RNase A, 9-14: different amounts of recombinant, bovine RNase A (from 1 μg to 0.025 μg) b) RNase A activity during the plasmid isolation

M: 1 kb size marker, 1: without RNase A, 2: bovine RNase A (company A), 3: bovine RNase A (company B), 4: recombinant RNase A

Claims

PATENT REPRESENTATION

1. A process for the production of recombinant RNase A in E. coli, characterized in that a DNA sequence is used which codes for an RNase A of bovine origin and which has been adapted to the codon use in E. coli.

2. The method of claim 1, wherein the DNA sequence to the

Codon usage in E. coli K12 is adapted.

3. The method of claim 1 or 2, wherein the DNA sequence is adapted to the codon most commonly used in E. coli.

4. The method according to any one of claims 1 to 3, wherein the DNA sequence of the SEQ ID. No. 1 specified DNA sequence or one to that in SEQ ID. No. 1 specified DNA sequence corresponds to at least 90% identical sequence.

5. The method according to claim 1 or 2, wherein the DNA sequence is adapted taking into account the natural frequency of individual codons.

6. The method according to any one of claims 1, 2 or 5, wherein the DNA sequence of the SEQ ID. No. 2 specified DNA sequence or one to that in SEQ ID. No. 2 specified DNA sequence corresponds to at least 90% identical sequence.

7. The method according to any one of the preceding claims, wherein the RNase A is expressed in fusion with a signal peptide which controls the transport into the periplasmic space.

8. The method of claim 7, wherein it sic-h in the signal peptide around the signal peptide of alkaline phosphatase (phoA) h-undelt.

9. The method according to any one of the preceding claims, wherein the expression of RNase A is under the control of an inducible promoter.

10. The method according to claim 9, wherein the promoter is a heat-inducible promoter.

11. The method according to spoke 9 or 10, wherein the induction of gene expression takes place at the end of the exponential growth phase.

12. The method according to any one of claims 9 to 11, wherein the induction of gene expression takes place for a period of 14 to 20 hours.

13. The method according to any one of the preceding claims, wherein the

RNase A Inclusion Bodies forms.

14. Method according to one of the preceding. Claims, the method further comprising obtaining the RNase A atus from the E. coli cells or the culture medium, if appropriate with solubilization and refolding of the RNase A.

15. The method according approached 14, wherein guanidine-HC1 is used as a denaturing agent for solubilization.

16. The method according to claim 14 or 15, wherein reduced and oxidized glutathione is used for refolding.

17. The method according to any one of the preceding claims, wherein the

The method further comprises a chromatographic purification of RNase A.

18. The method according to claim 17, wherein a cation exchange chromatography is carried out.

19. The method according to any one of the preceding claims, wherein the yield of RNase A is more than 100 mg RNase A per liter of culture medium.

20. The method according to any one of the preceding claims, wherein the yield of RNase A is more than 3 mg RNase A per gram of bio-moist mass.

21. Recombinant RNase A, produced by a process according to one of claims 1 to 20.

22. E. co / z ^' cell culture containing at least 0.2 g RNase A per liter

Culture medium.

23. Nucleic acid molecule containing a nucleic acid sequence according to SEQ ID No. 1.

24. Nucleic acid molecule containing a nucleic acid sequence according to SEQ ID No. Second

25. Nucleic acid molecule comprising the following components in 5 -3 'order:

a promoter active in E. coli,

- if necessary, a sequence coding for a signal peptide within the meaning of claim 7 or 8, - a nucleic acid sequence according to SEQ ID No. 1 or No. Second

26. Use of a nucleic acid sequence according to SEQ ID No. 1 or No. 2 for the production of recombinant RNase A.

27. Use of the RNase A according to claim 21 in the purification of

DNA and proteins.