Recombinant plasmid dna pestpc, encoding polypeptide with properties of esterase psychrobacter cryohalolentis k5t, escherichia coli bacteria strain - producer of polypeptide with properties of esterase psychrobacter cryohalolentis k5t

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology. Described is a plasmid which codes esterase Psychrobacter cryohalolentis K5T and contains Ndel/Xhol - a DNA fragment of plasmid pET32a with length of 5.366 thousand base pairs, which includes a promoter of bacteriophage T7, a translation enhancer of gene 10 of the bacteriophage T7, a transcription terminator of the bacteriophage T7, a bla β-lactamase gene which determines resistance of cells transformed by plasmid pPC023 to ampicillin, a replication initiation section ori; and Ndel/Sall - a DNA fragment with size of 0.869 thousand base pairs, which contains a gene which codes the mature form of esterase Psychrobacter cryohalolentis K5T with an amino acid sequence SEQ ID NO: 2, given in the description, having an C-end hexahestidine linker. Provided is an Escherichia coli bacteria strain which is modified by said plasmid, a producer of a polypeptide having esterase Psychrobacter cryohalolentis K5T activity.

EFFECT: invention increases the level of biosynthesis of esterase to 20% of total cell protein.

2 cl, 2 dwg, 5 ex


The scope of the invention

The invention relates to biotechnology, particularly genetic engineering. Can be used to obtain the enzyme esterasePsychrobacter cryohalolentisK5Tpossessing high catalytic activity at low temperatures in the reactions of hydrolysis of ester compounds. The esterasePsychrobacter cryohalolentisK5Tcan be used in the food industry for baking industry as additives to the test and in the manufacture of dairy products to accelerate the ripening of cheese; in light industry in the production of household chemicals as components of detergents and spot removers [Hasan, F., A. Shah, et al. Industrial applications of microbial lipases. Enzyme and Microbial Technology. 2006. V. 39(2). P. 235-251].

The level of technology

The main producers of holocausting of esterases are strains ofAspergillus, Fusarium, Mucor, Rhizopussome yeast (Candida) [Coenen, T., P. Aughton, et al. Safety evaluation of lipase derived from Rhizopus oryzae: Summary of toxicological data. Food and chemical toxicology. 1997. V. 35(3-4). P. 315-322; Zhang, N., W. C. Suen, et al. Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution. Protein engineering. 2003. V. 16(8). P. 599]. A method of obtaining esterase, based on the cultivation of strains of fungiFusarium oxysporum[P.Rapp. Production, regulation and some properties of lipase activity fromFusarium oxysporumf. sp.vasinfectum. Enzyme and Microbial Technology. 1995. V.17. P. 832-838]. When kultivirovaniya 30 º C for 150 hours maximum accumulation in culture liquid of specific esterase activity 150% act./mg of dry biomass. The disadvantage of this method is the duration of cultivation (6 - 7 days) and the complexity of the procedures for obtaining the culture fluid (exemption from mushroom mycelium).

A method of obtaining holocausting of esterases and lipases by culturing bacteria, such asSerratia marcescens[N. A. Bashkatova, LA Severin. Influence of cultivation conditions on the biosynthesis of lipase fromSerratia marcescens. Microbiology. 1979. So 68, Vol. 5. S. 826],Pseudomonas fluorescens[Dieckelmann, M., L. Johnson, et al. The diversity of lipases from psychrotrophic strains of Pseudomonas: a novel lipase from a highly lipolytic strain of Pseudomonas fluorescens. Journal of applied microbiology. 1998. V. 85(3). P. 527-536],Achromobacter lipolyticum[Khan, I. M., C. Dill, et al. Production and properties of the increasing interest among lipase ofAchromobacter lipolyticum.Biochimica et Biophysica Acta (BBA)-Enzymology. 1967. V. 132(1), p. 68-77]. The use of bacteria has compared with mushrooms advantage in speeding up the process of cultivation. Lack of receipt of esterases by culturing bacteria-producers is a difficult procedure of cleaning and low specific activity of the resulting enzyme.

Another known method of obtaining holocausting lipases and esterases is designing systems expressions of the genes of these enzymes inE. coliusing genetic engineering methods. An example is the system of lipase gene expression Lip1A psychrotrophic Mick is organizma Psychrobacter sp.[Zhang, J., S. Lin, et al. Cloning, expression, and characterization of a cold-adapted lipase gene from an antarctic deep-sea psychrotrophic bacterium,Psychrobacter sp.7195. J Environ Biotechnol. 2007. V. 17(4). P. 604-10] in cellsE. coliBL21(DE3)/ pUC-lipA1, providing access to 0.45 mg of protein from 1 l of culture. The disadvantage of this system is the low yield of the target protein, as well as difficult procedure of cleaning it.

Closest to the claimed method for producing a protein with esterase activity is a method of obtaining protein LipXHiswith lipase activity of psychrophilic bacteriaPsychrobacter sp.C18 [Chen, R., L. Guo, et al. Gene cloning, expression and characterization of a cold-adapted lipase from a psychrophilic deep-sea bacteriumPsychrobacter sp. C18. World Journal of Microbiology and Biotechnology. 2011. P. 1-11]. The recombinant plasmid pET28a(+)/lipX contains lipase genePsychrobacter sp.C18 under the control of the promoter of bacteriophage T7. Recombinant strain ofE. coliBL21(DE3)/ pET28a(+)/lipX provides protein synthesis LipXHiswith 6 histidine residues at the C-end, which allows its purification using Ni-affinity chromatography. Protein synthesis is carried out with the addition of the inducer isopropyl - β - D - thiogalactopyranoside (IPTG) to a final concentration of 0.2 mm. The result is a protein with lipase activityPsychrobacter sp.C18 with the release of 3.8 mg from 1 l of culture. The disadvantages of the method of obtaining protein LipXHiscan be attributed to the relatively low level of synthesis and not enough high specificactivity.

The invention

An object of the invention is to obtain a polypeptide with properties esterasePsychrobacter cryohalolentisK5Tand increase the level of its biosynthesis.

The problem is solved by constructing recombinant plasmid DNA pEstPc that encodes a polypeptide with esterase activity c mol. weight of 4.05 Md (6,235 KBP), consisting of NdeI/XhoI - fragment DNA plasmid pET32a(+) length 5,366 KBP, including the promoter of bacteriophage T7, the transcription terminator of bacteriophage T7 gene bla, β-lactamase, which determines the stability of the transformed plasmid pEstPc cells to ampicillin, plot ori of replication initiation; and NdeI/SalI - fragment DNA size 0,869 KBP containing the gene EstPc encoding the amino acid sequence of the Mature form of esterasePsychrobacter cryohalolentisK5T; containing a unique recognition sites of restriction endonucleases, with the following coordinates: NdeI - 367, XhoI - 840, and also due to the strain of bacteriaEscherichia coliBL21(DE3)/pEstPc producer polypeptide with properties esterasePsychrobacter cryohalolentisK5T.

Recombinant plasmid DNA pEstPc encodes the inducible synthesis of the polypeptide with properties esterasePsychrobacter cryohalolentisK5Tand the strain ofEscherichia coliBL21(DE3)/pEstPc provides a synthesis of this polypeptide with the level of expression of at least 20% of the total cell be is CA. Inducible high-level synthesis of the target polypeptide is provided that plasmid pEstPc contains the promoter of the bacteriophage T7 and the amplifier broadcast of the gene 10 of bacteriophage T7, and a deletion of the 5'end of the gene sequence of the esterasePsychrobacter cryohalolentisK5T,encodes a signal peptide.

A distinctive feature of the proposed plasmid constructions is the presence in its composition of the coding sequence of the Mature form of esterasePsychrobacter cryohalolentisK5T,devoid of the signal peptide (SEQ ID NO 1),under the control of a strong adjustable T7lac promoter, which, combined with the presence of plasmid gene repressor LacI and in the chromosome of the host strain gene RNA polymerase of bacteriophage T7, provides inducible synthesis of the target protein (SEQ ID NO 2) with reliable regulation and high yield, achieved at low concentrations of inducer. Previously when cloning full-esterase genePsychrobacter cryohalolentisK5Twe found that the expression level of this gene in cells ofEscherichia colivery low.The deletion of the fragment of the gene corresponding to the signal peptide, leads to increased levels of synthesis of the recombinant protein in the cytoplasm of bacterial cells. At the 3'end of a gene is a sequence encoding six histidine residues, to facilitate purification of the target white is and using metalroofing chromatography.

To obtain strain-producer of the polypeptide with properties esterasePsychrobacter cryohalolentisK5Tcompetent cellsEscherichia colistrain BL21(DE3) transformed with recombinant plasmid pEstPc.

The technical result of the claimed invention is that the resulting strainE. coliBL21(DE3)/pEstPc allows to obtain a polypeptide with properties esterasePsychrobacter cryohalolentisK5Tnot less than 20% of the total cellular protein at a concentration of inducer 0.1 mm. Unlike the prototype in this strain is achieved in 7.2 times higher yield of the target protein using 2 times less inductor (IPTG). The combination of the above-mentioned properties of the strain ofE. coliBL21(DE3)/pEstPc leads to increased manufacturability of the process of obtaining holocausting esterase.

List of figures

The invention is illustrated graphics.

Fig. 1. Physical map of recombinant plasmid pEstPc. Specified unique sites of restriction endonucleases. T7 promoter - the promoter of bacteriophage T7, T7 terminator - the terminator of transcription of bacteriophage T7, lacI gene lac-repressor, Ap - gene resistance to ampicillin, ori - the site of initiation of replication of plasmids, EstPc - recombinant gene EstPc encoding the amino acid sequence of the Mature form of esterasePsychrobacter cryohalolentisK5T.

Fig. 2. Electrophoregram of listblack producer strain E. coliBL21(DE3)/ pEstPc before (lane 1) and after induction (lane 2) in a 13%polyacrylamide gel (M - protein molecular weight markers); the arrow indicates the polypeptide with esterase activity.

Description. The resulting strainEscherichia coliBL21(DE3)/ pEstPc characterized by the following features.

Morphological signsthe cells are small, rod-shaped, gram-negative, motile, 1 × 3-5 µm, risperadone.

Cultural characteristicsfor the growth on agar LB-medium dominated by small colonies with a diameter of 1-3 mm; round, smooth, translucent, shiny, grey, edge smooth; the pasty consistency. Can meet large colonies with a diameter of 4-5 mm; round, smooth, Matt, edge wavy. Growth in liquid media (LB, minimal medium with glucose) is characterized by the clouding of the environment.

Physiological and biochemical characteristics.The cells of the producer strain can grow in the temperature range 20-42°C, the optimum temperature of growth is 37º. The most favorable for growth pH values are in the range of 6.8 to 7.2. When growing under aerobic conditions a culture can assimilate nitrogen as organic compounds (peptone, tripton, amino acids, yeast extract), and ammonium salts. Carbon is assimilated in the form of carbohydrates, polyhydric alcohols (glycerin), amino acids.

Stable is epostl to antibiotics. Cells are resistant to ampicillin (200 μg/ml), due to the presence of plasmid gene beta-lactamase.

The method, conditions and medium composition for the storage of strain.LB-broth with 15% glycerol at a temperature of - 70aboutIn cryovial.


The invention is illustrated by the following examples.

Example 1. Construction of recombinant plasmid DNA pEstPc.

CulturePsychrobacter cryohalolentisK5Tgrown over night on the Cup with agar medium TSB (Difco, USA)+2% NaCl. 20 μl of the obtained biomass suspended in 50 μl of buffer for Taq polymerase (75 mm Tris-HCl, pH of 8.8, 20 mm (NH4)2SO4, 0,1% Tween 20) and destroy within 15 min by heating in a boiling water bath. After centrifugation 5 min at a speed of 13000 rpm, 5 μl of the obtained supernatant added to the reaction mixture for PCR volume of 50 µl, containing 50 pmol of primer PC231F (SEQ ID NO 3), 50 pmol of primer PC231R (SEQ ID NO 4), 2 mm each of the four dNTP, 5 ál 10x buffer for Pfu DNA polymerase (Fermentas), 2.5 EA. Pfu DNA polymerase and 0.5 EA. Taq DNA polymerase (Fermentas). Polymerase chain reaction (PCR) is conducted as follows: denaturation 1 min, 94°C; annealing - 45 s, 52°C; completion - 45 s, 72°C; the number of cycles - 35. 10 µg of PCR product is treated with restrictase NdeI and SalI (Fermentas) under conditions recommended by the manufacturer of the enzymes, the fragment length 0,869 KBP allocate 1% agarose the first gel. 1 μg of the obtained fragment and 0.2 μg of plasmid vector pET32a(+), linearized by treatment with restrictase NdeI and XhoI, length BP 5366 are ligated for 4 h at 12°C in 10 µl of a solution containing 40 mm Tris-HCl (pH 7.8), 10 mm MgCl2,10 mm dithiothreitol, 0.5 mm ATP and 3 EA. T4 DNA ligase (Fermentas, Lithuania). 5 μl of ligase mixture used to transform competent cellsE. coliXL-1 Blue (Stratagene, USA). Transformants plated on LB-agar containing 100 μg/ml ampicillin.

From grown clones secrete plasmid DNA and treated with restrictase NdeI and XhoI, followed by electrophoretic analysis of the lengths of restriction fragments in a 1% agarose gel. The structure of the cloned gene in the selected clones was confirmed by determining the nucleotide sequence. The result is the target plasmid DNA pEstPc (Fig. 1).

Example 2. Obtaining strain-producer of the polypeptide with esterase activity.

Recombinant plasmid DNA pEstPc transform competent cells ofE. coliBL21(DE3) (Novagen) and after cultivation of recombinant clones on LB-agar with ampicillin (100 μg/ml) at 37aboutTo get producing strains polypeptide with esterase activity.

Example 3. Determination of productivity of the strain-producer of the polypeptide with esterase activity.

To determine the productivity of cellsE. coliBL21(DE3/pEstPc grow when 37º in 5 ml liquid LB medium, containing 100 μg/ml ampicillin, for 16 h on a shaker at 250 rpm/min Obtained nightlife culture in a volume of 100 μl is transferred into 5 ml of liquid LB medium (diluted to an optical density of 0.15-0.20 at a wavelength of 560 nm)containing 100 μg/ml ampicillin and grown to an optical density of 0.8 (wavelength 560 nm) at 37º and stirring of 250 rpm Add IPTG to a final concentration of 0.1 mm, after which the cells are grown for 4 h at 27º and stirring 200 rpm Selected sample culture in the quantity 1 optical unit (wavelength 560 nm) and centrifuged 5 min at a speed of 7000 rpm Precipitated cells suspended in 100 μl of buffer containing 125 mm Tris-HCl, pH of 6.8, 20% glycerol, 3% sodium dodecyl sulphate, 3% mercaptoethanol, of 0.005% bromophenol blue, incubated for 10 min in a boiling water bath, the sample volume of 10 µl analyzed by electrophoresis in a 13% polyacrylamide gel with sodium dodecyl sulfate. After electrophoresis the gel stain using Kumasi R-250. After washing the dye gel scan and carry out mathematical processing of the results using the program Scion Image (Scion Corp., USA). According to scan the contents of esterasePsychrobacter cryohalolentis20% of the total cellular protein. Figure 2 presents electrophoregram lysates of cells of strain-producerE. coliBL21(DE3)/pEsyPc (lanes 1 and 3) in a 13%polyacrylamide is the first gel before (lane 1) and after induction IPTG (track 2) (M - protein molecular weight markers; the arrow indicates the polypeptide with esterase activity).

Example 4. Isolation and purification of the recombinant esterasePsychrobacter cryohalolentis.

Spend the induction of esterase biosynthesisPsychrobacter cryohalolentisin cells of strain-producerE. coliBL21(DE3)/pEstPc IPTG (final concentration 0.1 mm) for 4 h, the Cells centrifuged 15 min at 7000 rpm

1 g of wet biomass induced cells resuspended in 10 ml of buffer (50 mm Tris-HCl pH 8.0; 200 mm NaCl), after which destroy cells by treatment in an ultrasonic disintegrator Branson Sonifier 450, not allowing the temperature more than 10°C. the resulting suspension is centrifuged 15 min at 17000 rpm isolation and purification of the desired polypeptide of the supernatant is performed using Ni-affinity chromatography on a column of Ni-Sepharose FastFlow (GE Healthcare) with a volume of 2 ml in the concentration gradient of imidazole (0.1 - 0.5 M) in buffer (20 mm Tris-HCl pH an 8.0; 200 mm NaCl). Combined fractions containing purified protein and having data on protein electrophoresis purity more than 90%, dialist against buffer (20 mm Tris-HCl pH 8.0; 50 mm NaCl) and sterilized. The average yield of purified so esterase is 28 mg from 1 l of culture, which is 7 times greater than that of the prototype.

Example 5. The determination of the activity of recombinant esterasePsychrobacter cryohalolentisK5T

The esterase activity determined using paranitro the Nile butyrate (Sigma) as substrate [Kulakova, L., A. Galkin, et al. Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly-->Pro substitution near the active site on its catalytic activity and stability. Biochim Biophys Acta. 2004. V.1696(1). P. 59-65]. To the reaction mixture in a volume of 1 ml containing 50 mm Tris-HCl (pH 8.0), 100 mm NaCl, 0,5% Triton X100, 0,25 mm paranitrophenyl of butyrate (Sigma), add 1,5 µg purified protein, the mixture is incubated for 15 min at 30ºC. 200 μl of the reaction mixture after centrifugation at 13,000 rpm for 5 min digging into the wells of 96-hole tablet (Costar). Measured value of the absorbance at 415 nm on a plate reader Model680 (BioRad). Per unit esterase activity take the amount of enzyme required to release 1 µmol of paranitrophenol for 1 min Specific activity is defined as the number of units esterase activity per 1 mg of protein. General esterna activity of the purified target polypeptide in comparison with the prototype 15 times more, and the specific activity is 1300 edict./mg protein, which is 2 times higher than the specific activity of the prototype.

1. Recombinant plasmid DNA RRs encoding a polypeptide with the activity of esterase Psychrobacter cryohalolentisK5Twith mol. weight of 4.05 Md (6,235 KBP), consisting of NdeI/XhoI - fragment DNA plasmids RETA length 5,366 KBP, including the promoter of bacteriophage T7, power broadcast 10 gene bacterial plankton the Aga T7, the transcription terminator of bacteriophage T7 gene bla, β-lactamase, which determines the stability of the transformed plasmid RRs cells to ampicillin, plot ori of replication initiation; and NdeI/SalI - fragment DNA size 0,869 KBP containing the gene Rsquo encoding the amino acid sequence of SEQ ID NO:2 Mature forms of esterase Psychrobacter cryohalolentis K5Twith C-end exegetically linker.

2. The bacterial strain Escherichia coli BL21(DE3)/pPC023 producing polypeptide with the activity of esterase Psychrobacter cryohalolentis K5T.


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