CN117363596A - Mutant ribonuclease R and application thereof - Google Patents
Mutant ribonuclease R and application thereof Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
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Abstract
The invention provides a mutant ribonuclease R (RNase R) and application thereof. The invention provides 10 mutant ribonuclease R through creatively selecting and screening the RNase R site mutation of the escherichia coli, and the mutant T495E, A246E, K457F, A246F, D281F, D122L with obviously improved relative to the parent enzyme activity is obtained. Wherein the mutant T495E has the most obvious enzyme activity improvement, the relative enzyme activity is improved by 37.48 percent, and the enzyme activities of other mutants are respectively improved by 24.74 percent, 21.74 percent, 10.04 percent, 10.34 percent and 6.75 percent. The invention also provides recombinant escherichia coli for high expression of RNase R, which is used for realizing efficient production of RNase R mutants and has good industrial application prospect. The ribonuclease R mutant with improved enzyme activity provided by the invention is suitable for large-scale industrial mRNA preparation, and is particularly applied to the fields related to degradation of linear RNA or production of circRNA.
Description
Technical Field
The invention relates to the fields of genetic engineering and medicines, in particular to the field of ribonuclease R, and especially relates to mutant ribonuclease R and application thereof. The application is a divisional application with the application number of 202211610204.8 and the name of 'mutant ribonuclease R and application thereof'.
Background
Ribonuclease R (RNase R) is a magnesium-dependent 3 '. Fwdarw.5 ' exoribonuclease that digests substantially all linear RNA but does not digest lasso-like or circular RNA structures, or double stranded RNA 7 nucleotides shorter than the 3' overhang. Most cellular RNA will be completely digested by RNase R, except tRNA,5S RNA and integrins. The 3' -tail of the lasso will be modified by the RNase R to a branch point nucleotide in which a 2',5' -phosphodiester linkage is present.
Circular RNAs (circrnas) are a new class of Circular non-coding RNAs that differ from linear RNAs, with long half-life, species conservation and tissue specificity. The unique ring structure of the polypeptide is not easy to degrade by RNase, so that the polypeptide has strong intracellular stability and great potential and research value in the directions of novel biomarkers, biological mechanism research and the like.
The concept of "circRNA" was first found in 1971 in RNA viruses, but has long been recognized as "noise" of transcription because of its low abundance of expression, and has no practical effect. Until 2012, salzman et al (Salzman J, gawad C, wang P L, et al, circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types, plos One,2012,7 (2): 1-12) studied that there were non-linear circular transcripts formed by exon rearrangement, whether leukemia cells, hela cell lines or normal human primary blood cells, were first reported. Subsequent large numbers of researchers have been surmounted into this area and continue to produce research results on circular RNAs. The most studied at present is circular RNA formed by exons located in cytoplasm, which contains a large number of miRNA binding sites and can play a role of miRNA sponge, bind and seal the regulation of miRNA, thereby enhancing the expression of target genes.
With the intensive research of circular RNAs, the problem of circular RNA production is becoming more and more interesting. In the preparation process of the circular RNA, in order to eliminate the interference of other RNA, RNase R is required to eliminate other RNA in a reaction system so as to obtain purer circRNA. However, the quality of the RNase R existing at present is greatly different, and certain RNase R is found to have obvious degradation effect on the circRNA in the verification process. With the increasing heat of research on circular RNAs, the demand for RNase R will increase further in the future market and research fields.
CN114438054A (publication date 2022.05.06) discloses a mutant RNase R designated as RNase R-. DELTA.M8, the amino acid sequence of which is shown in SEQ ID NO.5, and the nucleotide sequence encoding the amino acid sequence of which is shown in SEQ ID NO. 6. The preparation process of the mutant RNase R delta M8 provided by the invention comprises the processes of vector construction, vector transformation, protein induction expression, bacterial collection, protein purification, activity measurement and the like. Compared with the wild-type RNase R, the mutant RNase R provided by the invention has higher protein expression amount and can tolerate 150mM NaCl.
Although mutant RNase R's are known in the art, there remains a need in the art to provide different, performance enhancing mutant RNase R's for industrial and scientific research, particularly mutant RNase R's with enhanced enzymatic activity, to obtain higher yields and purity of circRNA.
Disclosure of Invention
In view of the drawbacks of the prior art in the field of RNase R, it is an object of the present invention to provide a mutant RNase R with improved properties, in particular significantly improved enzymatic activity relative to the parent. The invention further aims to provide a recombinant strain for high-expression of RNase R, in particular to recombinant escherichia coli, which is used for realizing high-efficiency production of RNase R mutants and has good industrial application prospect. The invention also provides a recombinant nucleic acid for coding the mutant RNase R, an expression cassette, a vector, a cell, a strain, a composition and a kit containing the nucleic acid, a preparation method, a purification method and an enzyme activity detection method of the RNase R mutant, and application of the RNase R mutant in the fields of degrading linear RNA, producing circRNA and the like.
In one aspect, the invention provides a mutant ribonuclease R, which is characterized by comprising a mutation based on a parent ribonuclease R, wherein the mutation comprises T495E, A246E, K457F, A246F, D281F, or D122L, and the amino acid sequence of the parent ribonuclease R is shown as SEQ ID NO. 1.
Further, the mutant ribonuclease R is mutant T495E, A246E, K457F, A246F, D281F or D122L, the amino acid sequence of the mutant T495E is shown as SEQ ID NO. 30, the amino acid sequence of the mutant A246E is shown as SEQ ID NO. 25, the amino acid sequence of the mutant K457F is shown as SEQ ID NO. 29, the amino acid sequence of the mutant A246F is shown as SEQ ID NO. 26, the amino acid sequence of the mutant D281F is shown as SEQ ID NO. 27, and the amino acid sequence of the mutant D122L is shown as SEQ ID NO. 23.
Further, the DNA sequence of the parent ribonuclease R is shown as SEQ ID NO. 2.
In another aspect, the invention provides a recombinant nucleic acid comprising a nucleic acid encoding said mutated ribonuclease R.
In another aspect, the invention provides an expression cassette comprising the recombinant nucleic acid.
In another aspect, the invention provides a vector comprising said recombinant nucleic acid or said expression cassette; preferably, the vector is an expression vector.
In another aspect the invention provides a cell comprising said recombinant nucleic acid or said expression cassette or said vector.
In another aspect, the present invention provides a recombinant bacterium comprising the recombinant nucleic acid or the expression cassette or the vector; preferably, the bacterium is escherichia coli; more preferably, the escherichia coli is one of e.coli BL21 (DE 3), e.coli Origami B (DE 3) or e.coli Rosetta Blue (DE 3); most preferably, the E.coli is E.coli BL21 (DE 3).
In another aspect the invention provides a composition characterized in that it comprises said mutated ribonuclease R or said recombinant nucleic acid or said expression cassette or said vector or said cell or said recombinant bacterium.
In another aspect the invention provides a kit characterized in that it comprises said mutated ribonuclease R or said recombinant nucleic acid or said expression cassette or said vector or said cell or said recombinant bacterium or said composition.
In another aspect, the present invention provides a method for preparing the mutant ribonuclease R, comprising the steps of:
(1) Constructing a vector containing a nucleotide sequence encoding said mutated ribonuclease R;
(2) Transforming the vector obtained in the step (1) into cells of an expression strain to obtain the expression strain;
(3) Performing amplification culture on the expression strain obtained in the step (2) and performing protein induction expression;
(4) Collecting the expression strain after the expansion culture, and washing and cracking;
(5) Purifying the protein;
(6) And (5) performing enzyme activity detection.
Further, the step (3) comprises culturing the expression strain obtained in the step (2) at 25-37 ℃ to OD 600 And adding isopropyl-beta-D-thiogalactoside (IPTG) to a culture medium with a final concentration of 0.1-1.5mM, and performing induced fermentation at 16-37 ℃ for 8-16h to obtain the mutant ribonuclease R.
Further, the step (4) comprises centrifuging the fermentation broth obtained in the step (3) at 8000rpm for 5min and discarding the supernatant, then re-suspending the cells with 50mM PBS and centrifuging at 8000rpm for 5min and discarding the supernatant, adding PBS to re-suspend the cells, and mixing uniformly; sonicating cells on ice; the cell disruption solution was placed in a pre-chilled centrifuge, centrifuged at 8000rpm for 10min at 4℃to collect the pellet, and the pellet was reconstituted in KCl buffer containing 500 mM.
In another aspect, the present invention provides a method for purifying the mutant ribonuclease R, comprising the steps of: and (3) purifying the crude enzyme solution containing the mutant ribonuclease R obtained by fermentation by using a protein purification system, balancing a nickel column by using a balancing solution in advance, eluting by using an eluent with increasing concentration, collecting target proteins, and then performing electrophoresis analysis and detection.
In another aspect, the present invention provides a method for detecting the enzymatic activity of the mutant ribonuclease R, comprising the steps of: the mutant ribonuclease (RNase R) was diluted by a series of gradients, the diluted RNase R was degraded at 37℃for 10. Mu.g of linear mRNA, and after 1h, the detection was performed by gel electrophoresis until no distinct bands were detected by electrophoresis, and the enzyme activity of the mutant ribonuclease R was calculated from the amount of enzyme without distinct bands.
In another aspect, the invention provides the use of said mutated ribonuclease R in the field of mRNA preparation.
In another aspect, the invention provides the use of said mutant ribonuclease R in the field of degrading linear RNA or producing circRNA.
The mutant ribonuclease R and the application thereof provided by the invention have the following beneficial technical effects:
1. the invention provides 10 mutant ribonuclease R through creatively selecting and screening the RNase R site mutation of the escherichia coli, and obtains mutant T495E, A246E, K457F, A246F, D281F, D122L with obviously improved relative to the parent enzyme activity. Wherein the mutant T495E has the most obvious improvement of enzyme activity, and the relative enzyme activity is improved by 37.48 percent. The second position is mutant A246E, and the relative enzyme activity is improved by 24.74%. The third position is mutant K457F, and the relative enzyme activity is improved by 21.74%. The relative enzyme activity of the mutant A246F, D281F is improved by about 10 percent. The relative enzyme activity of the mutant D122L is improved by less than 10 percent and is 6.75 percent.
2. The method for preparing the mutant RNase R is simple and quick to operate, and the obtained enzyme has good stability.
3. The invention provides a recombinant strain for high-expression RNase R, in particular recombinant escherichia coli, which is used for realizing high-efficiency production of RNase R mutants and has good industrial application prospect.
4. Compared with the parent, the mutant ribonuclease R provided by the invention has obviously improved enzyme activity, is particularly suitable for large-scale industrial mRNA preparation, and is especially applied to the fields related to degradation of linear RNA or production of the circRNA, and the circRNA with higher yield and purity can be obtained.
Drawings
FIG. 1 is a map of pET-28a-RNase R vector of the present invention, wherein RNase R is Feature 15 in the map;
FIG. 2 shows SDS-PAGE detection results of expressed RNase R whole cells (whole bacteria) and expression amounts of supernatant (soluble) proteins of a disruption solution of recombinant E.coli BL21 (DE 3) of the present invention under different IPTG final concentrations;
FIG. 3 is a chart showing a nickel column chromatography procedure of an RNase R-containing crude enzyme solution expressed by Escherichia coli according to the present invention;
FIG. 4 shows SDS-PAGE results of protein content of off-peak components collected during different time periods of the nickel column chromatography of the present invention;
FIG. 5 shows the results of electrophoresis of linear EGFP mRNA remaining after degradation of RNase R at different dilutions according to the present invention.
Detailed Description
EXAMPLE 1 construction of parent RNase R expression vector
Through genome analysis of escherichia coli, the gene is found to contain ribonuclease RNase R, and the protein sequence encoded by the gene contains 814 amino acid residues, and belongs to the RNR superfamily. The amino acid sequence of RNase R protein is shown as SEQ ID NO.1, codon optimization is carried out according to the expression of escherichia coli, and finally the obtained DNA sequence is shown as SEQ ID NO.2, and the specific sequence is shown as table 1. The DNA sequence was submitted to the company (Tianzhin Biolimited) and the gene was successfully constructed on the pET-28a vector to obtain pET-28a-RNase R, the plasmid expression pattern of which is shown in FIG. 1.
TABLE 1 parental RNase R sequence information
EXAMPLE 2 E.coli inducible expression of ribonuclease RNase R
E.coli JM109 strain containing the synthesized pET-28a-RNase R of example 1 was inoculated into LB liquid medium containing 50mg/L kanamycin for cultivation, followed by extraction of the plasmid to obtain recombinant plasmid pET-28a-RNase R. The recombinant expression vector pET-28a-RNase R is transformed into competent cell E.coli BL21 (DE 3) for positive clone selection.
The recombinant strain E.coli BL21 (DE 3) obtained by screening is induced to express by IPTG. Inoculating the recombinant strain into LB culture medium, culturing at 37deg.C to OD 600 1.8-2.2, then, the cell culture broth was centrifuged to collect the cells at 8000g for 5min, then, LB medium was added to resuspend the cells, and the OD of the cells was resuspended 600 0.9-1.1, and simultaneously adding IPTG to the culture medium to a final concentration of 0.5mM, and performing induced fermentation at 25deg.C for 8 hr. Centrifuging, collecting bacterial precipitate, ultrasonic crushing, and performing SDS-PAGE electrophoresis analysis, wherein the molecular weight of the target protein RNase R is about 92kDa. The SDS-PAGE detection results of expressed ribonuclease RNase R whole cells (whole bacteria) and the expression amount of broken supernatant (soluble) protein of recombinant E.coli BL21 (DE 3) under different IPTG final concentrations are shown in figure 2, the lanes of the electrophoresis chart are, from left to right, 1 lane Marker,2 lane BL21-pET28 a+RNase-R-1-25-0 IPTG whole bacteria, 3 lane BL21-pET28 a+RNase-R-1-25-0.1% IPTG soluble, 4 lane BL21-pET28 a+RNase-R-1-25-0.1% IPTG whole bacteria, 5 lane BL21-pET28 a+RNase-R-1-25% to 0.1% IPTG is soluble, 6 BL21-pET28 a+RNase-R-1-25% to 0.5% IPTG whole bacteria, 7 BL21-pET28 a+RNase-R-1-25% to 0.5% IPTG is soluble, 8 BL21-pET28 a+RNase-R-1-25% to 1.0% IPTG whole bacteria, 9 BL21-pET28 a+RNase-R-1-25% to 1.0% IPTG is soluble, and-1 of RNase-R-1 in the lanes represents the number of the monoclonal strain. The results show that the target protein RNase R has better expression under the condition (the final concentration of IPTG is 0.5mM, corresponding to lane 6, namely 0.5 percent of IPTG), and can be used for preparing thalli by batch fermentation.
EXAMPLE 3RNase R Nickel column purification and gel column desalting
When recombinant escherichia coli BL21 (DE 3)/pET 28a-RNase R induces and expresses proteins, the target proteins are finally distributed in cells, and the cells are firstly crushed to obtain the target proteins. The fermentation broth was poured into a 50ml centrifuge tube, centrifuged at 8000rpm for 5min and the supernatant was discarded, after which the cells were resuspended in 50mM PBS and centrifuged at 8000rpm for 5min and the supernatant was discarded, and PBS was added to resuspend the cells, which were then homogenized by pipetting. In the process of ultrasonic cell disruption, thalli should be placed on ice, and the conditions of an ultrasonic cell disruptor are set as follows: horn 6mm, time 60min,50ml of cell suspension and ensure that the probe is close to but not touching the bottom. The cell disruption solution was placed in a pre-chilled centrifuge, centrifuged at 8000rpm for 10min at 4℃to collect the pellet, and the pellet was reconstituted in KCl buffer containing 500 mM. Subsequently, insoluble impurities in the sample were filtered off using a 0.45 μm filter (Sidoris manufacturer) before the crude enzyme sample was purified.
E.coli contains RNase R crude enzyme solution for purification. The RNase R crude enzyme solution was subjected to Ni column (Nami, 77105-60042-531800) purification using AKTA protein purification system. Balance: a Ni-NTA column previously equilibrated with Buffer A (50mM NaH2PO4,0.5M NaCl,pH 8.0) was used, buffer A (50 mM NaH) 2 PO 4 0.5M NaCl, pH 8.0) rinse 5CV (column volume), load after all baseline equilibrates: and (3) taking a KCl buffer solution, re-dissolving, passing through a membrane, and loading the sample, and collecting a flow-through sample. Washing: balancing solution Buffer A (50 mM NaH) 2 PO 4 0.5M NaCl, pH 8.0) to baseline, pH, conductivity plateau. Eluting: buffer B (50 mM NaH) 2 PO 4 0.5M NaCl,0.5M imidazole, pH 8.0) eluent 0-50% gradient elution, washing volume 30CV, collecting sample peaks, and reserving sample for detection. Eluting: 100% buffer B (50 mM NaH) 2 PO 4 0.5M NaCl,0.5M imidazole, pH 8.0) eluate was washed 5CV and monitored at 280nm, and sample peaks were collected and detected as a leave-on. A nickel column chromatography program chart of the RNase R-containing crude enzyme solution expressed by the Escherichia coli is shown in FIG. 3.
Purified samples were tested by SDS-PAGE. The sample is mixed with protein Loading Buffer (SDS, DTT and bromophenol blue) in a ratio such that the final sample has a protein Loading Buffer concentration of not less than 1X,98℃or a boiling water bath for 10min. Subsequently, an electrophoresis tank and a rubber plate (sealing-removing adhesive tape) are installed, electrophoresis buffer solution is poured into the electrophoresis tank, the inner tank should overflow, and the liquid level of the outer tank should be higher than that of the electrode wire. Slowly removing the comb of the prefabricated glue, taking the needle cylinder to absorb the buffer solution, and gradually extending into the glue holes to blow off the glycerol. Sample application, closing the tank cover, electrifying, electrophoresis for 40 minutes under 80V voltage, and compressing the sample into a line. The electrophoresis was continued by increasing the voltage value by 120V until the indicator tape was near or reached the bottom of the gel. Dyeing and decoloring the gel after electrophoresis by using a protein dyeing instrument, and setting the program to be Stain 3min; destin 2min; destin 1min. The gel is transferred into a gel imaging system, imaged, image modified, lanes arranged, lanes marked and corresponding analysis is performed. SDS-PAGE results of the peak component protein content collected by the nickel column chromatography for different time periods are shown in FIG. 4, wherein the lanes of the electrophoresis chart are sequentially from left to right, 1 lane LC1A0130 μl,2 lane RNase-R-2 30 μl,3 lane marker,4 lane XT1A07 30 μl,5 lane XT1A08 μl,6 lane XT1A09 μl,7 lane XT1A11 μl,8 lane XT1A12 μl,9 lane XT1B12 μl,10 lane XT1B11 μl, and the-2 of RNase-R-2 in the lanes represents the monoclonal strain number. The results show that: the eluted target protein RNase R can be purified by a one-step nickel column to obtain more purified products.
The purified sample was passed through a gel column (nano-micro, 77105-60011-003400) to remove imidazole. Mixing the protein sample containing the target protein, which is obtained by eluting the nickel column, and taking the mixture as a sample loaded on a gel column. And removing imidazole from the purified enzyme solution by using an AKTA protein purification system. Balance: using a gel column pre-equilibrated with Buffer a, buffer a was used to rinse 5CV with all baseline equilibrated, and loaded: and loading the sample purified by the nickel column, collecting the flow-through sample, stopping collecting after the conductivity rises, then flushing the gel column by using a Buffer A, loading again after all base lines are balanced until all the samples are subjected to imidazole removal treatment, and then carrying out ultrafiltration concentration on the collected sample by using an ultrafiltration tube to obtain RNase R enzyme solution.
Example 4 enzyme Activity detection of RNase R enzyme solution
And (3) enzyme activity detection of RNase R enzyme solution. Defining the RNase R enzyme activity; in 20mM Tris-HCl (pH 7.5), 100mM KCl,0.5mM Mg 2+ Under conditions, the amount of enzyme required to hydrolyze 1. Mu.g of linear mRNA product at 37℃is 1 active unit. As an example of the sample (not limited to the linear EGFP mRNA sample, any linear mRNA sample may be used), the prepared RNase R was subjected to a series of gradient dilutions, the diluted RNase R was degraded at 37℃to 10. Mu.g of linear EGFP mRNA, and after 1 hour, the residual linear EGFP mRNA after degradation was detected by gel electrophoresis, and the results of electrophoresis of the different gradient dilutions of the RNase R were shown in FIG. 5, with lanes of the electrophoresis chart starting from left to right, 1 lane DL 5000marker,2 lanes of control (control, i.e., linear EGFP mRNA without RNase R added), 3 lanes of 0.01. Mu.L RNase R-mRNA,4 lanes of 0.05. Mu.L RNase R-mRNA,5 lanes of 0.1. Mu.L RNase R-mRNA,6 lanes of 0.15. Mu.Nase R-mRNA,7 lanes of 0.2. Mu.L RNase R-mRNA,8 lanes of 0.25. Mu.L RNase R-mRNA, 9. Mu.L of RNase R-mRNA, and lanes 3 to 9 refer to linear EGFP mRNA samples. The enzyme solution added with 0.25 mu L is catalyzed for 1h at 37 ℃,10 mu g of mRNA can be completely degraded until no obvious band is formed in final running, and finally the enzyme activity of the prepared parent enzyme RNase R is 6.67U/. Mu.L according to the dosage of the final enzyme.
EXAMPLE 5 construction of RNase R mutant and enzyme Activity detection of enzyme solution for enzyme Activity detection
5.1 construction of E.coli ribonuclease RNase R mutant
Primers directed to the parent D122L, K205L, A E, A246F, D281F, D281Y, K457F, T495E, L610W, V687N mutation (lower case mutation base) were designed and synthesized respectively according to the parent enzyme RNase R gene sequence (DNA nucleotide sequence shown in SEQ ID NO: 2) amplified in example 1 using PCR technique, and the primers used for PCR are shown in Table 2. The amino acid sequences of the RNase R mutants are shown in Table 3. The amino acid sequence of RNase R mutant D122L is shown as SEQ ID NO. 23, the amino acid sequence of mutant K205L is shown as SEQ ID NO. 24, the amino acid sequence of mutant A246E is shown as SEQ ID NO. 25, the amino acid sequence of mutant A246F is shown as SEQ ID NO. 26, the amino acid sequence of mutant D281F is shown as SEQ ID NO. 27, the amino acid sequence of mutant D281Y is shown as SEQ ID NO. 28, the amino acid sequence of mutant K457F is shown as SEQ ID NO. 29, the amino acid sequence of mutant T495E is shown as SEQ ID NO. 30, the amino acid sequence of mutant L610W is shown as SEQ ID NO. 31, and the amino acid sequence of mutant V687N is shown as SEQ ID NO. 32.
The PCR reaction system is as follows: 2. Mu.L of forward primer (10. Mu.M), 2. Mu.L of reverse primer (10. Mu.M), 1. Mu.L of template DNA, 25. Mu.L of NEBNext Ultra IIQ Master Mix (manufacturer NEB, 10135601) and double distilled water were added to 50. Mu.L.
The PCR amplification conditions were: pre-denaturation at 98℃for 3min; followed by 30 cycles (98 ℃ C. 10s,55 ℃ C. 15s,72 ℃ C. 5 min); the extension was continued for 10min at 72 ℃. After the PCR product is verified to be correct, the PCR product is digested by DpnI and then is transformed into competent E.coli Tans10, competent cells are cultured in LB solid medium (containing 50mg/L kana) overnight, plasmids are extracted after being selected and cloned in LB liquid medium containing 50mg/L kana, mutant plasmids are transformed into competent cells of expression host E.coli BL21 (DE 3), and all mutant plasmids are sequenced correctly. Recombinant strains were obtained, named D122L, K205L, A246E, A F, D281F, D281Y, K457F, T495E, L610W, V687N, respectively.
TABLE 2 construction of primer list by RNase R mutant
TABLE 3 amino acid sequence of RNase R mutant
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5.2 expression and purification of E.coli ribonuclease RNase R mutant
The RNase R mutant expression engineering strain constructed in the step 5.1 was subjected to the expression of the mutant RNase R according to example 2 and the purification of the mutant RNase R according to example 3, and finally purified RNase R mutant enzyme was obtained.
5.3 detection of enzyme Activity of E.coli ribonuclease RNase R mutant
The enzyme activities of the mutants were examined according to the method of example 4, and the degradation activities of the wild-type parent E.coli RNase R (WT) and the mutants on mRNA are shown in Table 4, and the results indicate that the enzyme activities of 10 ribonuclease RNase R mutants, D122L, K205L, A246E, A246F, D F, D281 457 495E, L610W, V687N, were increased by 6.75%, 8.28%, 24.74%, 10.04%, 10.34%, 4.95%, 21.74%, 37.48%, 23.24%, and 8.1% respectively (where negative values represent decreases) relative to the wild-type. From this, the enzyme activity of the T495E mutant in the 10 RNase R mutants is improved most significantly, and the relative enzyme activity is improved by 37.48%. The second position is mutant A246E, and the relative enzyme activity is improved by 24.74%. The third position is mutant K457F, and the relative enzyme activity is improved by 21.74%. The relative enzyme activity of the mutant A246F, D281F is improved by about 10 percent. The relative enzyme activity of the mutant D122L is improved by less than 10 percent and is 6.75 percent. The effect on mutant K205L, D281Y, L610W, V687N is poor, and the relative enzyme activity is reduced to different degrees. Wherein, for D281F, D281Y, the different types of mutation at the same mutation site have different effects, the relative enzyme activity of D281F is improved, and the relative enzyme activity of D281Y is reduced. Thus, the mutant T495E, A246E, K457F, A246F, D281F, D122L with obviously improved relative to the parent enzyme activity is obtained by creatively selecting and screening the RNase R locus of the escherichia coli ribonuclease and the amino acid type after mutation.
TABLE 4 relative enzyme Activity of wild-type E.coli RNase R and multiple mutant enzymes
The above examples of the present disclosure are merely examples for clearly illustrating the present disclosure and are not limiting of the embodiments of the present disclosure. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modifications, equivalent substitutions, improvements, etc. that fall within the spirit and principles of the present disclosure are intended to be included within the scope of the claims of the present disclosure.
Claims (21)
1. The mutant ribonuclease R is characterized by comprising mutation on the basis of a parent ribonuclease R, wherein the mutation is D122L, and the amino acid sequence of the parent ribonuclease R is shown as SEQ ID NO. 1.
2. The mutant ribonuclease R of claim 1, wherein the mutant ribonuclease R is mutant D122L, and the amino acid sequence of the mutant D122L is shown in SEQ ID NO. 23.
3. The mutant ribonuclease R of claim 1 or 2, wherein the DNA sequence of the parent ribonuclease R is as set forth in SEQ ID No. 2.
4. A recombinant nucleic acid comprising a nucleic acid encoding the mutated ribonuclease R of any one of claims 1-3.
5. An expression cassette comprising the recombinant nucleic acid of claim 4.
6. A vector comprising the recombinant nucleic acid of claim 4 or the expression cassette of claim 5.
7. The vector of claim 6, wherein the vector is an expression vector.
8. A cell comprising the recombinant nucleic acid of claim 4, or the expression cassette of claim 5, or the vector of claim 6 or 7.
9. A recombinant bacterium comprising the recombinant nucleic acid of claim 4, or the expression cassette of claim 5, or the vector of claim 6 or 7.
10. The recombinant bacterium according to claim 9, wherein the bacterium is escherichia coli.
11. The recombinant bacterium according to claim 10, wherein the escherichia coli is one of e.coli BL21 (DE 3), e.coli Origami B (DE 3) or e.coli Rosetta Blue (DE 3).
12. The recombinant bacterium according to claim 11, wherein the escherichia coli is e.coli BL21 (DE 3).
13. A composition comprising a mutant ribonuclease R according to any one of claims 1 to 3, a recombinant nucleic acid according to claim 4, an expression cassette according to claim 5, a vector according to claim 6 or 7, a cell according to claim 8, or a recombinant bacterium according to any one of claims 9 to 12.
14. A kit comprising a mutant ribonuclease R according to any one of claims 1 to 3, a recombinant nucleic acid according to claim 4, an expression cassette according to claim 5, a vector according to claim 6 or 7, a cell according to claim 8, a recombinant bacterium according to any one of claims 9 to 12, or a composition according to claim 13.
15. A method for preparing the mutant ribonuclease R of any one of claims 1-3, comprising the steps of:
(1) Constructing a vector containing a nucleotide sequence encoding said mutated ribonuclease R;
(2) Transforming the vector obtained in the step (1) into cells of an expression strain to obtain the expression strain;
(3) Performing amplification culture on the expression strain obtained in the step (2) and performing protein induction expression;
(4) Collecting the expression strain after the expansion culture, and washing and cracking;
(5) Purifying the protein;
(6) And (5) performing enzyme activity detection.
16. The method according to claim 15, wherein the step (3) comprises culturing the expression strain obtained in the step (2) at 25 to 37℃to OD 600 And adding isopropyl-beta-D-thiogalactoside (IPTG) to a culture medium with a final concentration of 0.1-1.5mM, and performing induced fermentation at 16-37 ℃ for 8-16h to obtain the mutant ribonuclease R.
17. The method according to claim 15 or 16, wherein step (4) comprises centrifuging the fermentation broth obtained in step (3) at 8000rpm for 5min and discarding the supernatant, then resuspending the cells with 50mM PBS and centrifuging at 8000rpm for 5min and discarding the supernatant, adding PBS and resuspending the cells, and mixing; sonicating cells on ice; the cell disruption solution was placed in a pre-chilled centrifuge, centrifuged at 8000rpm for 10min at 4℃to collect the pellet, and the pellet was reconstituted in KCl buffer containing 500 mM.
18. A method for purifying a mutant ribonuclease R according to any one of claims 1 to 3, comprising the steps of: and (3) purifying the crude enzyme solution containing the mutant ribonuclease R obtained by fermentation by using a protein purification system, eluting by using a nickel column which is balanced by a balancing solution in advance and an eluent with increasing concentration, collecting target proteins, and then carrying out electrophoresis analysis and detection.
19. A method for detecting the enzymatic activity of the mutant ribonuclease R according to any one of claims 1 to 3, comprising the steps of: and (3) diluting the mutant ribonuclease R through a series of gradients, degrading 10 mug of linear mRNA by the diluted mutant ribonuclease R at 37 ℃, detecting by gel electrophoresis after 1h until no obvious band is detected by electrophoresis, and calculating the enzyme activity of the mutant ribonuclease R according to the use amount of the enzyme without the obvious band.
20. Use of a mutated ribonuclease R according to any one of claims 1 to 3 in the field of mRNA preparation.
21. Use of a mutated ribonuclease R according to any one of claims 1 to 3 in the field of degradation of linear RNA or production of circRNA.
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