RU2773954C1 - Method for producing recombinant protein of serratia marcescens bacteria nuclease for hydrolysis of nucleic acids - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, specifically, to recombinant production of enzymes in E. coli, and can be used to produce recombinant protein of Serratia marcescens nuclease. The method includes producing an oligonucleotide duplex encoding the corresponding gene SEQ ID NO: 2 S. marcescens, optimised for expression in E. coli; creating a plasmid containing the sequence of the gene SEQ ID NO: 2 of S. marcescens nuclease, and producing a producer strain; growing producer cells of the E. coli strain, expressing the gene of S. marcescens nuclease without a signal sequence, in an inactive, non-toxic and insoluble form - as inclusion bodies within bacterial cells; destructing the created producer cells, isolating, washing the inclusion bodies from strain proteins to produce the finished product; further regenerating the resulting Serratia marcescens nuclease to the native conformation, and purifying.

EFFECT: invention provides a high level of nuclease biosynthesis in E. coli, allows for a reduction in the time of the process cycle of nuclease purification with an increase in the output of the target protein.

1 cl, 6 ex

Description

The present invention relates to microbiology and biotechnology and consists in creating a recombinant nuclease preparation from a Gram-negative bacterium Serratia marcescens. The creation of the drug includes the expression of the nuclease in the cytoplasm of Escherichia coli cells in an insoluble form in the form of "inclusion bodies", the subsequent refolding of the native conformation of the enzyme and the method of its purification to a homogeneous state.

As is known, nucleases, hydrolytic enzymes that cleave nucleic acids, are of great economic importance. Non-specific nucleases are mainly used for the hydrolysis of nucleic acids in various processes. A nuclease that cleaves only DNA is called a DNase, while a nuclease that cleaves only RNA is called an RNase. A typical representative of DNases is DNase I from the mammalian pancreas. Representative RNases are, for example, RNases T1 and T2 from Aspergillus oryzae or RNase A from mammalian pancreas.

In addition to purification of DNA or RNA with RNases or DNases, respectively, there are other applications of economic interest in which the sample is purified from both nucleic acids. This applies, for example, to the production of a wide range of molecules of protein or non-protein origin, in which the product does not consist of nucleic acids: proteins such as antibodies or enzymes; polysaccharides; lipids; antibiotics, etc. The need to remove nucleic acids becomes a particularly important task if the production of molecules occurs intracellularly or if some of the producing cells are lysed during production. As a result, during the preparation of the produced substance, large amounts of nucleic acids are present in the preparation, which contaminate the produced substance or make it difficult to further purify it. Purification is difficult, among other things, because the nucleic acids increase the viscosity of the preparations to such an extent that subsequent steps such as filtration or chromatography become impossible.

Therefore, there is interest in methods for the complete or partial removal of nucleic acids. Such methods can remove manufacturing restrictions at further stages of substance development and exclude the presence of nucleic acids in pharmaceutical preparations. One approach for removing nucleic acids is to precipitate nucleic acids with various agents. Another approach is to cleave nucleic acids by nucleases into sufficiently small fragments. This approach makes it possible to reduce the viscosity of the samples, and the resulting hydrolysis products can be subsequently removed using available methods, such as ultrafiltration or column chromatography.

The use of nucleases, which can cleave both RNA and DNA, is particularly useful for removing all kinds of nucleic acids, i.e. both RNA and DNA, from various samples. In this case, the nuclease used should have high activity and sufficient stability. The enzyme that exhibits these properties is the nuclease of the Gram-negative bacterium S. marcescens [EC 3.1.30.2; SEQ ID NO: 1; Filimonova M. N. et al. Biochemistry, 1980, 45 (11): 2096-2104; Filimonova M. N. et al. Biochemistry, 1981, 46 (9): 1660-1666; Biedermann K et al. Res commun. 1989, 54 (1): 17-27]. This enzyme is also sold under the brand name Benzonase [Molin S et al. US 5,173,418 Dec. 22, 1992] and hereinafter referred to as "S. marcescens nuclease".

There are numerous technical solutions related to the biosynthesis of nuclease by Serratia marcescens bacteria (Ed. St. USSR No. 230052, 1967; Ed. St. USSR No. 340691, 1970; Ed. St. USSR No. 992568; patent RU 2665550) .

The disadvantage of the developments described above is the use of Serratia marcescens as a producer strain. This producer is a conditionally pathogenic organism and requires special cultivation conditions, nutrient composition, and the use of the antibiotic mitomycin C as an inducer.

In order to be able to economically produce proteins in sufficient quantities and with the required purity, they are often obtained using industrial organisms by the method of heterologous expression, i.e. DNA encoding the gene of the protein of interest is introduced into the producing organism, which then carries out the expression, i.e. synthesis of a foreign protein. This approach can have a number of advantages: increased productivity of the producer organism compared to the original organism, more optimized processes of cultivation, development and purification of the product.

Nucleases can have a toxic effect on the producing organism if expressed intracellularly. In the cytoplasm, an active nuclease hydrolyzes the nucleic acids of the host cell, which can lead to cell death or inhibition of cell growth. Violation of secretion can also lead to the death or growth retardation of the producer.

Recombinant secretory expression of S. marcescens nuclease in the gram-negative bacterium Escherichia coli is described in [Molin S et al. EP 229866 B1 1992] SEQ 10 NO1 and is presented in comparison with the expression yield in the wild strain - S. marcescens W225. It was shown that in the expression system used, the nuclease yield was 35,000 units/ml of the culture obtained with the recombinant E. coli strain and 7,000 units/ml with the original strain. Moreover, it has been shown that approximately half of the enzymatic activity remains in the periplasm of E. coli and is not secreted into the medium (Table 4 in EP 229866 B1 1992). Biedermann K et al. (Appl. Environ. Microbiol. 1990, 56(6): 1833-1838) also describe the secretion of S. marcescens nuclease (from S. marcescens strain W280 SEQ ID NO1) in E. coli cells that synthesize it. The study shows a comparison of the rate of nuclease secretion in a homologous gram-negative host organism S. marcescens and a similar gram-negative model organism E. coli. The yield of nuclease per ml of culture liquid under fermentation conditions of the producer strain E. coli is 17,000 units/ml.

Gram-negative bacteria in general, and E. coli in particular, have some disadvantages. On the one hand, secretion is often only possible in small yields and is usually carried out only into the periplasm and not directly into the medium, making subsequent purification difficult. In this variant, it is necessary to process large volumes of preparations obtained from the bacterial periplasm.

An invention is also known in which, with heterologous expression of S. marcescens nuclease in a gram-positive host of Bacillus subtilis [Greiner-Stoeffele T and Schoenert S US 9,796,994 B2 2017), the nuclease yield is 7,200 units/ml of culture fluid.

The objective of the claimed technical solution is to eliminate the shortcomings of the prototypes, namely, the development of a method for the biosynthesis of S. E. marcescens nuclease in coli cells, which allows to achieve an increase in the biosynthesis of S. marcescens nuclease under the action of an isopropyl-beta-O-thiogalactopyranoside (IPTG) inducer using standard media and technologies developed for E. coli. The essence of the invention consists in creating a non-pathogenic S. marcescens nuclease producer expressing the gene of this enzyme without a signal sequence in an inactive, non-toxic and insoluble form in the form of inclusion bodies in the cytoplasm of E. coli cells and obtaining a preparation of a recombinant S. marcescens nuclease protein refolded to a native conformation , which is easy to clean and has a high specific activity. Another technical problem to be solved by the claimed group of inventions is the development of a drug based on the specified protein suitable for use in biotechnological practice. It should be noted that the amino acid sequence of the recombinant protein of this invention corresponds to the mature form of S. marcescens nuclease with the amino acid sequence of SEQ ID NO: 2, which corresponds to the sequence of SEQ ID NO: 1 without the nuclease transport signal of SEQ ID NO: 3 SEQ ID NO: 2 contains additional amino acid residues (in bold) for translation initiation and genetic manipulation (restriction endonuclease sites introduced for cloning).

The specified technical result is achieved by obtaining a recombinant protein with a molecular weight of 27 kDa SEQ ID NO: 2, including a fragment of the S. marcescens nuclease protein SEQ ID NO: 1 without the signal sequence of SEQ ID NO: 3.

The technical result is also achieved by a method for obtaining a recombinant S. marcescens nuclease protein, which includes:

- growing E. coli cells expressing the S. marcescens nuclease gene without a signal sequence in an inactive and non-toxic form - in the form of inclusion bodies inside bacterial cells;

- destruction of E. coli strain cells, isolation and washing of inclusion bodies from strain proteins;

- dissolution of inclusion bodies in urea and subsequent dialysis procedure - refolding of Serratia marcescens nuclease to native conformation;

- two-stage chromatographic purification of S. marcescens nuclease from side proteins.

Thus, an E. coli strain was created that expresses the Serratia marcescens nuclease gene without a signal sequence in an inactive, insoluble and non-toxic form - in the form of inclusion bodies in the cytoplasm of a bacterial cell, a procedure for refolding Serratia marcescens nuclease to a native state and a highly efficient procedure for purifying Serratia marcescens nuclease from side proteins.

The claimed technical solution is explained in the sequence listing, which presents variants of the amino acid sequence of the S. marcescens nuclease.

List of sequences explaining the essence of the invention:

SEQ ID NO: 1 shows the complete amino acid sequence of S. marcescens nuclease, the signal sequence is underlined.

SEQ ID NO: 2 shows the amino acid sequence of the mature form of S. marcescens nuclease without the signal sequence.

SEQ ID NO: 3 shows the amino acid sequence of the S. marcescens nuclease signal sequence.

a fragment of the S. marcescens nuclease protein without a signal sequence, followed by its cloning.

The S. marcescens nuclease gene was obtained by a chemoenzymatic method. Was created oligonucleotide duplex encoding the corresponding gene, optimized for expression in E. coli. Then the pNucA plasmid containing the sequence of the S. marcescens nuclease gene was obtained.

Example 2. Obtaining a strain of E. coli - producer.

To obtain an E. coli strain producing the recombinant S. marcescens nuclease protein, E. coli BL21 cells were transformed with the pNucA plasmid. 3 μl of 0.1 M IPTG solution was added to the culture and grown for 3 hours at 37°C. When comparing the spectrum of proteins synthesized by the cells of the E. coli BL21 [pNucA] strain with the proteins of the untransformed strain, the appearance of an additional protein band was revealed. The molecular weight of the additional band corresponded to that expected for the recombinant S. marcescens nuclease protein, 27 kDa. The level of protein synthesis in E. coli was determined by comparing the intensity of staining of the band of the recombinant protein with the band of the corresponding protein - standard molecular weight. It has been shown that the recombinant S. marcescens nuclease protein is synthesized in E. coli cells in an insoluble form as inclusion bodies. The level of target protein synthesis was 10% of the total protein of the bacterial cell.

Example 3 Washing of inclusion bodies and refolding of recombinant S. marcescens nuclease

The destruction of the cells of the producer strain was carried out under standard conditions with 50 mM Tris-HCl, pH 8.0, 0.25 M NaCl 0.5% on a French press. The inclusion bodies were centrifuged at 10,000 rpm, 30 min, 10°C. The inclusion bodies were washed in lysis buffer (3 times), sedimenting by centrifugation in the same mode.

Inclusion bodies were dissolved in buffer: 8 M urea, 20 mM Tris-HCl, pH 8.0, 0.1% Triton X-100. Insoluble components were removed by centrifugation at 10,000 rpm, 30 min, 20°C. The inclusion body solution was dialyzed by reducing the concentration of urea from 8 M to 0 M in 25 mM sodium acetate buffer pH 5.3. Insoluble components were removed by centrifugation at 10,000 rpm, 30 min, 10°C.

Example 4 Cleaning on WorkBeads 40 S ion exchange resin

Chromatography on a WorkBeads 40 S sorbent column was performed in 25 mM sodium acetate, pH 5.3. The target protein was eluted with a NaCl gradient from 0 to 1 M in 25 mM sodium acetate, pH 5.3. The resulting protein fractions were analyzed by electrophoresis in 12% SDS-PAGE according to Laemmli. The protein concentration was determined spectrophotometrically by the Bradford method. Protein-containing fractions were dialyzed against 20 mM Tris-HCl, pH 8.0 with double buffer change at 4°C.

Example 5 Cleaning on WorkBeads 40 DEAE Ion Exchange Resin

Chromatography on a column with WorkBeads 40 DEAE sorbent was carried out in 20 mM Tris-HCl buffer, pH 8.0. The sorbent with protein immobilized on it is washed with 20 mM Tris-HCl buffer, pH 8.0 until the UV detector signal drops and stabilizes. The target protein was eluted with a NaCl gradient from 0 to 0.5 M in 20 mM Tris-HCl, pH 8.0.

The resulting protein fractions were analyzed by electrophoresis in 12% SDS-PAGE according to Laemmli. The protein concentration was determined spectrophotometrically by the Bradford method. The purified protein was dialyzed against 20 mM Tris-HCl buffer, pH 8.0.

The dialyzed protein solution was filtered into sterile vials through a 0.20 µm filter. The protein concentration was determined spectrophotometrically by the Bradford method.

Example 6 Measurement of DNase Activity of S. marcescens Nuclease

To measure the DNase activity of the S. marcescens nuclease, the method of acid-soluble fractions was taken as the basis [Leshchinskaya I.B. and others. Biochemistry, 1974, T. 39, No. 1, S. 116-122]. The reaction mixture for determining the activity of S. marcescens nuclease under standard conditions contained a 50 mM Tris-HCl buffer, pH 8.0; 2 mm MgSO ₄ and 0.1% high molecular weight DNA.

The reaction mixture was prepared by mixing in test tubes previously placed in an ice bath. The test enzyme was added to the prepared mixture in a volume ratio of 1:9 in various dilutions, and hydrolysis was carried out in a thermostat for 15 min. at 37°C. After that, the hydrolysis was stopped, the mixture was placed in an ice bath, and an equal volume of 4% perchloric acid was added to the mixture. The amount of low molecular weight fragments formed as a result of DNA hydrolysis was determined and DNase activity was calculated as described in the original method [Leshchinskaya I.B. and others. Biochemistry, 1974, T. 39, No. 1, S. 116-122]. As a test reference drug, Benzonase from Merck USA (prototype) and S. marcescens nuclease according to this invention, previously diluted with a 20 mM Tris-HCl buffer, pH 8.0, were used so that no more than 30-50% of DNA was converted into hydrolyzed fragments. In parallel with the test solutions, distilled water was added to the reaction mixtures instead of these solutions, and, having performed all similar manipulations with the test samples, DNA hydrolysis was evaluated.

The DNase activity of the nuclease of S. marcescens strain E. coli BL21 [pNucA] according to this invention was 48500 units/ml of culture fluid. Comparison with other developments: EP 229866 B1 - 35000 U/ml, Biedermann K et al. - 17000 u/ml, US 9,796,994 B2 - 7200 u/ml shows that the level achieved exceeds that previously described in other inventions and articles.

The achieved purity of the S. marcescens nuclease preparation according to the present invention was 99%. Specific activity - 250-300 units/µl. The nuclease preparation remained active for a year when stored in 50% glycerol at (-) 20°C.

Thus, the proposed development demonstrated that a recombinant protein - S. marcescens nuclease without a signal sequence, can be synthesized in E. coli bacteria in an inactive, insoluble and non-toxic form in the form of "inclusion bodies". This technical solution provides a high level of nuclease biosynthesis by E. coli bacteria. The use of standard procedures for refolding and chromatographic purification makes it possible to obtain a preparation of appropriate purity suitable for biotechnological practice. The achieved purity of the S. marcescens nuclease preparation according to the present invention was 99%. Specific activity - 250-300 units/µl. It should be noted that the created nuclease preparation retained its activity for a year with a certain storage. The production method also makes it possible to reduce the time of the technological cycle of nuclease purification while increasing the yield of the target protein. This development can be applied in applied microbiology, biotechnology, as well as in the microbiological and pharmaceutical industries.

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Sequence listing

<210> 1

<211> 266

<212> PRT

<213> Serratia marcescens

<400> 1

Met Arg Phe Asn Asn Lys Met Leu Ala Leu Ala Ala Leu Leu Phe Ala

1 5 10 15

Ala Gln Ala Ser Ala Asp Thr Leu Glu Ser Ile Asp Asn Cys Ala Val

20 25 30

Gly Cys Pro Thr Gly Gly Ser Ser Asn Val Ser Ile Val Arg His Ala

35 40 45

Tyr Thr Leu Asn Asn Asn Ser Thr Thr Lys Phe Ala Asn Trp Val Ala

50 55 60

Tyr His Ile Thr Lys Asp Thr Pro Ala Ser Gly Lys Thr Arg Asn Trp

65 70 75 80

Lys Thr Asp Pro Ala Leu Asn Pro Ala Asp Thr Leu Ala Pro Ala Asp

85 90 95

Tyr Thr Gly Ala Asn Ala Ala Leu Lys Val Asp Arg Gly His Gln Ala

100 105 110

Pro Leu Ala Ser Leu Ala Gly Val Ser Asp Trp Glu Ser Leu Asn Tyr

115 120 125

Leu Ser Asn Ile Thr Pro Gln Lys Ser Asp Leu Asn Gln Gly Ala Trp

130 135 140

Ala Arg Leu Glu Asp Gln Glu Arg Lys Leu Ile Asp Arg Ala Asp Ile

145 150 155 160

Ser Ser Val Tyr Thr Val Thr Gly Pro Leu Tyr Glu Arg Asp Met Gly

165 170 175

Lys Leu Pro Gly Thr Gln Lys Ala His Thr Ile Pro Ser Ala Tyr Trp

180 185 190

Lys Val Ile Phe Ile Asn Asn Ser Pro Ala Val Asn His Tyr Ala Ala

195 200 205

Phe Leu Phe Asp Gln Asn Thr Pro Lys Gly Ala Asp Phe Cys Gln Phe

210 215 220

Arg Val Thr Val Asp Glu Ile Glu Lys Arg Thr Gly Leu Ile Ile Trp

225 230 235 240

Ala Gly Leu Pro Asp Asp Val Gln Ala Ser Leu Lys Ser Lys Pro Gly

245 250 255

Val Leu Pro Glu Leu Met Gly Cys Lys Asn

260 265

<210> 2

<211> 248

<212> PRT

<213> Serratia marcescens

<400> 2

Met Gly Ser Asp Thr Leu Glu Ser Ile Asp Asn Cys Ala Val Gly Cys

1 5 10 15

Pro Thr Gly Gly Ser Ser Asn Val Ser Ile Val Arg His Ala Tyr Thr

20 25 30

Leu Asn Asn Asn Ser Thr Thr Lys Phe Ala Asn Trp Val Ala Tyr His

35 40 45

Ile Thr Lys Asp Thr Pro Ala Ser Gly Lys Thr Arg Asn Trp Lys Thr

50 55 60

Asp Pro Ala Leu Asn Pro Ala Asp Thr Leu Ala Pro Ala Asp Tyr Thr

65 70 75 80

Gly Ala Asn Ala Ala Leu Lys Val Asp Arg Gly His Gln Ala Pro Leu

85 90 95

Ala Ser Leu Ala Gly Val Ser Asp Trp Glu Ser Leu Asn Tyr Leu Ser

100 105 110

Asn Ile Thr Pro Gln Lys Ser Asp Leu Asn Gln Gly Ala Trp Ala Arg

115 120 125

Leu Glu Asp Gln Glu Arg Lys Leu Ile Asp Arg Ala Asp Ile Ser Ser

130 135 140

Val Tyr Thr Val Thr Gly Pro Leu Tyr Glu Arg Asp Met Gly Lys Leu

145 150 155 160

Pro Gly Thr Gln Lys Ala His Thr Ile Pro Ser Ala Tyr Trp Lys Val

165 170 175

Ile Phe Ile Asn Asn Ser Pro Ala Val Asn His Tyr Ala Ala Phe Leu

180 185 190

Phe Asp Gln Asn Thr Pro Lys Gly Ala Asp Phe Cys Gln Phe Arg Val

195 200 205

Thr Val Asp Glu Ile Glu Lys Arg Thr Gly Leu Ile Ile Trp Ala Gly

210 215 220

Leu Pro Asp Asp Val Gln Ala Ser Leu Lys Ser Lys Pro Gly Val Leu

225 230 235 240

Pro Glu Leu Met Gly Cys Lys Asn

245

<210> 3

<211> 11

<212> PRT

<213> Serratia marcescens

<400> 3

10 20

Met Arg Phe Asn Asn Lys Met Leu Ala Leu Ala Ala Leu Leu Phe Ala

1 5 10 15

Ala Gln Ala Ser Ala

twenty

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Claims (6)

A method for producing a recombinant S. marcescens nuclease protein with a molecular weight of 27 kDa, based on the expression of a gene encoding SEQ ID NO: 2 of S. marcescens nuclease without a signal sequence in an E. coli producer cell, which makes it possible to obtain the final product for hydrolysis of nucleic acids with a purity of 99 % and specific activity - 250-300 units/µl and including: - obtaining an oligonucleotide duplex encoding the corresponding gene SEQ ID NO: 2 S. marcescens, optimized for expression in E. coli; - creating a plasmid containing the sequence of the gene SEQ ID NO: 2 nuclease S. marcescens, and obtaining a producer strain; - growing cells of E. coli strain producers expressing the S. marcescens nuclease gene without a signal sequence, in an inactive, non-toxic and insoluble form - in the form of inclusion bodies inside bacterial cells; - destruction of the created producer cells, isolation, washing of inclusion bodies from strain proteins to obtain the final product; - subsequent recovery of the resulting Serratia marcescens nuclease to native conformation and purification.