CN116240199B - Mutant ribonuclease R and application thereof - Google Patents

Mutant ribonuclease R and application thereof Download PDF

Info

Publication number
CN116240199B
CN116240199B CN202211610204.8A CN202211610204A CN116240199B CN 116240199 B CN116240199 B CN 116240199B CN 202211610204 A CN202211610204 A CN 202211610204A CN 116240199 B CN116240199 B CN 116240199B
Authority
CN
China
Prior art keywords
mutant
ribonuclease
rnase
vector
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202211610204.8A
Other languages
Chinese (zh)
Other versions
CN116240199A (en
Inventor
左涛
刘立
汪芸
王鹏源
王满朝
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jiangsu Yao Hai Biopharmaceutical Co ltd
Original Assignee
Jiangsu Yao Hai Biopharmaceutical Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jiangsu Yao Hai Biopharmaceutical Co ltd filed Critical Jiangsu Yao Hai Biopharmaceutical Co ltd
Priority to CN202311371859.9A priority Critical patent/CN117327678A/en
Priority to CN202311371640.9A priority patent/CN117363596A/en
Priority to CN202311372194.3A priority patent/CN117402854A/en
Priority to CN202311371681.8A priority patent/CN117210438A/en
Priority to CN202211610204.8A priority patent/CN116240199B/en
Publication of CN116240199A publication Critical patent/CN116240199A/en
Application granted granted Critical
Publication of CN116240199B publication Critical patent/CN116240199B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/16Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22Ribonucleases RNAses, DNAses
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70Vectors or expression systems specially adapted for E. coli
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00Preparation of compounds containing saccharide radicals
    • C12P19/26Preparation of nitrogen-containing carbohydrates
    • C12P19/28N-glycosides
    • C12P19/30Nucleotides
    • C12P19/34Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RINDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00Microorganisms ; Processes using microorganisms
    • C12R2001/01Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185Escherichia
    • C12R2001/19Escherichia coli
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/914Hydrolases (3)
    • G01N2333/916Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)
    • G01N2333/922Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)

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

Mutant ribonuclease R and application thereof
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.
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 the presence of circular transcripts forming non-linearities by exon rearrangement, whether leukemia cells, hela cell lines or normal human primary blood cells, was 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 named RNase R-. DELTA.M8, whose amino acid sequence is shown as SEQ ID NO. 5 and the nucleotide sequence encoding the amino acid sequence is shown as 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 E.coli isE. coli BL21(DE3)、E. coli Origami B (DE 3) orE. coli One of Rosetta Blue (DE 3); most preferably, the E.coli isE. coli BL21(DE3)。
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 isopropyl-beta-D-thiogalactoside (IPTG) is added into a culture medium to a final concentration of 0.1-1.5 mM, and the fermentation is induced at 16-37 ℃ for 8-16 h, so that the mutant ribonuclease R can be induced and expressed.
Further, the step (4) comprises centrifuging the fermentation broth obtained in the step (3) at 8000 rpm for 5 min and discarding the supernatant, then re-suspending the cells with 50mM PBS and centrifuging at 8000 rpm for 5 min 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 8000 rpm for 10 min 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 to 10. Mu.g of linear mRNA at 37℃and detected by gel electrophoresis after 1 h 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 recombinant E.coli of the present inventionE. coli BL21 (DE 3) expressed RNase R whole cell (whole bacterium) under different IPTG final concentrationsAnd SDS-PAGE detection results of the expression quantity of the supernatant (soluble) protein of the crushed liquid;
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
Coli containing synthetic pET-28a-RNase R of example 1E. coli JM109 strain 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. Transformation of recombinant expression vector pET-28a-RNase R into competent cellsE. coli In BL21 (DE 3), positive clone selection was performed.
Recombinant strains obtained by screeningE. coli BL21 (DE 3) was expressed by IPTG induction. Inoculating the recombinant strain into LB culture medium, culturing at 37deg.C to OD 600 1.8-2.2, centrifuging the cell culture solution to collect thalli under 8000 g for 5 min, adding LB culture medium to resuspend the cells, and resuspending cell OD 600 0.9-1.1, while IPTG was added to the medium to a final concentration of 0.5 mM, and fermentation was induced at 25℃for 8 h. Centrifuging, collecting bacterial precipitate, ultrasonic crushing, and performing SDS-PAGE electrophoresis analysis, wherein the molecular weight of the target protein RNase R is about 92 kDa. Recombinant E.coliE. coli The SDS-PAGE detection results of expressed RNase R whole cells (whole bacteria) and the supernatant (soluble) protein expression amount of the crushing liquid of BL21 (DE 3) under different IPTG final concentrations are shown in figure 2, the lanes of the electrophoresis chart are sequentially from left to right, lane 1 Marker, lane 2 BL21-pET28a+RNase-R-1-25 ℃ to 0 IPTG whole bacteria, lane 3 BL21-pET28a+RNase-R-1-25 ℃ to 0.1% IPTG soluble, lane 4 BL21-pET28a+RNase-R-1-25 ℃ to 0.1% IPTG whole bacteria, lane BL21-pET28a+RNase-R-1-25 ℃ C. -0.1% IPTG is soluble, lane BL21-pET28a+RNase-R-1-25 ℃ C. -0.5% IPTG whole bacteria, lane BL21-pET28a+RNase-R-1-25 ℃ C. -0.5% IPTG is soluble, lane BL21-pET28a+RNase-R-1-25 ℃ C. -1.0% IPTG whole bacteria, lane BL21-pET28a+RNase-R-1-25 ℃ C. -1.0% IPTG is soluble, and lane RNase-R-1 represents the monoclonal strain number. The results show that the target protein RNase R has better expression under the condition (the final concentration of IPTG is 0.5 mM, corresponding to lane 6, namely 0.5 percent of IPTG), and can be used for preparing thalli by batch fermentation.
EXAMPLE 3 RNase 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. Pouring the fermentation broth into a 50 ml centrifuge tube, centrifuging at 8000 rpm for 5 min and discarding the supernatant, then re-suspending the cells with 50mM PBS and centrifuging at 8000 rpm for 5 min and discarding the supernatant, adding PBS to re-suspend the cells, and blowing uniformly with a pipette. 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 6 mm, time 60 min,50 mL of cell suspension and ensure that the probe is near but not touching the bottom. The cell disruption solution was placed in a pre-chilled centrifuge, centrifuged at 8000 rpm for 10 min 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 (50 mM NaH2PO4, 0.5M NaCl,pH 8.0) was used, buffer A (50 mM NaH) 2 PO 4 0.5M NaCl,pH 8.0) is rinsed 5 CV (column volume) and loaded after all baselines are balanced: 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) rinse 10 CV 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 30 CV, collecting sample peaks, and sample retention detection. Eluting: 100% Buffer B (50 mM NaH) 2 PO 4 0.5M NaCl, 0.5M imidazole, pH 8.0) eluent washes 5 CV and monitors at 280 nm, collecting sample peaks, and leaving behind for detection. 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 proportion to make the concentration of protein Loading Buffer of the final sample not lower than 1X, 98 ℃ or 10 min of boiling water bath denaturation. 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, and electrophoresis under 80-V voltage for 40 minutes until the sample is compressed into a line. The electrophoresis was continued by increasing the voltage value 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 3 min; destin 2 min; destin 1 min. 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 LC1A 01. Mu.l, 2 lane RNase-R-2 30. Mu.l, 3 lane marker,4 lane XT1A07 30. Mu.l, 5 lane XT1A 08. Mu.l, 6 lane XT1A 09. Mu.l, 7 lane XT1A 11. Mu.l, 8 lane XT1A 12. Mu.l, 9 lane XT1B 12. Mu.l, 10 lane XT1B 11. Mu.l, and-2 of RNase-R-2 in the lanes represents the serial numbers of the monoclonal strains. 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 washed 5 CV 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 20 mM Tris-HCl (pH 7.5), 100 mM KCl,0.5 mM Mg 2+ The amount of enzyme required to hydrolyze 1. Mu.g of linear mRNA product at 37℃under conditions was 1 active unit. Take linear EGFP mRNA as sample example (not limited to linear EGFP mRNA sampleAny linear mRNA sample is possible), the prepared RNase R is subjected to serial gradient dilution, the diluted RNase R is degraded into 10 mu g of linear EGFP mRNA at 37 ℃,1 h is detected by gel electrophoresis, the electrophoresis results of the residual linear EGFP mRNA after the degradation of the RNase R with different gradient dilutions are shown in FIG. 5, the electrophoresis patterns are shown in sequence from left to right, 1 lane DL 5000 marker,2 lane control (the control is the linear EGFP mRNA without the RNase R), 3 lane 0.01 mu L of RNase R-mRNA,4 lane 0.05 mu L of RNase R-mRNA,5 lane 0.1 mu L of RNase R-mRNA,6 lane 0.15 mu L of RNase R-mRNA,7 lane 0.2 mu L of RNase R-mRNA,8 lane 0.25 mu L of RNase R-mRNA,9 lane 0.3 mu L of RNase R-mRNA, and mRNA in lanes 3-9 refer to linear EGFP mRNA samples. The enzyme solution added with 0.25 mu L catalyzes 1 h at 37 ℃, so that 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 use amount 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 II Q 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 3 min; followed by 30 cycles (98 ℃ C. For 10 s,55 ℃ C. For 15 s,72 ℃ C. For 5 min); the extension was continued for 10 min 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, the 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
Primer name Primer sequence (5 '-3')
D122L-F CGTGTATTCATGGCcttCAGGTACTGGCTC(SEQ ID NO:3)
D122L-R GAGCCAGTACCTGaagGCCATGAATACACG(SEQ ID NO:4)
K205L-F CGTCCTACCCGTCGCACTcttGCTGTGGGCAAAATC(SEQ ID NO:5)
K205L-R GATTTTGCCCACAGCaagAGTGCGACGGGTAGGACG(SEQ ID NO:6)
A246E-F GCGGTTGAGCAACAAGTTgaaGGATTAAAAGAGGAAGTAC(SEQ ID NO:7)
A246E-R GTACTTCCTCTTTTAATCCttcAACTTGTTGCTCAACCGC(SEQ ID NO:8)
A246F-F CACAGGCGGTTGAGCAACAAGTTtttGGATTAAAAGAGGAAGTAC(SEQ ID NO:9)
A246F-R GTACTTCCTCTTTTAATCCaaaAACTTGTTGCTCAACCGCCTGTG(SEQ ID NO:10)
D281F-F GAGGATGCGCGTGACTTCGATtttGCAGTTTACTGTGAGAAAAAAC(SEQ ID NO:11)
D281F-R GTTTTTTCTCACAGTAAACTGCaaaATCGAAGTCACGCGCATCCTC(SEQ ID NO:12)
D281Y-F GATGCGCGTGACTTCGATtatGCAGTTTACTGTGAGAAAAAAC(SEQ ID NO:13)
D281Y-R GTTTTTTCTCACAGTAAACTGCataATCGAAGTCACGCGCATC(SEQ ID NO:14)
K457F-F CACAGCGCAACGACGCACATtttCTGATCGAAGAGTGCATGATC(SEQ ID NO:15)
K457F-R GATCATGCACTCTTCGATCAGaaaATGTGCGTCGTTGCGCTGTG(SEQ ID NO:16)
T495E-F GATCATGCACTCTTCGATCAGaaaATGTGCGTCGTTGCGCTGTG(SEQ ID NO:17)
T495E-R GATCATGCACTCTTCGATCAGaaaATGTGCGTCGTTGCGCTGTG(SEQ ID NO:18)
L610W-F CACTACTCAATGGAGGAAATGtggCAGCTGGGACAGCATTGC(SEQ ID NO:19)
L610W-R GCAATGCTGTCCCAGCTGccaCATTTCCTCCATTGAGTAGTG(SEQ ID NO:20)
V687N-F GACTACTACCGCTTCGATCAAaatGGGCAACGTTTAATGGG(SEQ ID NO:21)
V687N-R CCCATTAAACGTTGCCCattTTGATCGAAGCGGTAGTAGTC(SEQ ID NO:22)
TABLE 3 amino acid sequence of RNase R mutant
RNase R mutation Body Amino acid sequence
D122L MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGL RRRLRAMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYL SSEQMKTCIHGLQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFV VPDDSRLSFDILIPPDQIMGARMGFVVVVELTQRPTRRTKAVGKIVEVLGDNMGT GMAVDIALRTHEIPYIWPQAVEQQVAGLKEEVPEEAKAGRVDLRDLPLVTIDGED ARDFDDAVYCEKKRGGGWRLWVAIADVSYYVRPSTPLDREARNRGTSVYFPSQVI PMLPEVLSNGLCSLNPQVDRLCMVCEMTVSSKGRLTGYKFYEAVMSSHARLTYTK VWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKAREERGGISFESEEAKFIFNA ERRIERIEQTQRNDAHKLIEECMILANISAARFVEKAKEPALFRIHDKPSTEAIT SFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLLRSMKQAIYDP ENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETGGYHYS MEEMLQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFF VRLDDLFIDGLVHVSSLDNDYYRFDQVGQRLMGESSGQTYRLGDRVEVRVEAVNM DERKIDFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFR GEKKTKPKAAKKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE*(SEQ ID NO:23)
K205L MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGL RRRLRAMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYL SSEQMKTCIHGDQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFV VPDDSRLSFDILIPPDQIMGARMGFVVVVELTQRPTRRTLAVGKIVEVLGDNMGT GMAVDIALRTHEIPYIWPQAVEQQVAGLKEEVPEEAKAGRVDLRDLPLVTIDGED ARDFDDAVYCEKKRGGGWRLWVAIADVSYYVRPSTPLDREARNRGTSVYFPSQVI PMLPEVLSNGLCSLNPQVDRLCMVCEMTVSSKGRLTGYKFYEAVMSSHARLTYTK VWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKAREERGGISFESEEAKFIFNA ERRIERIEQTQRNDAHKLIEECMILANISAARFVEKAKEPALFRIHDKPSTEAIT SFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLLRSMKQAIYDP ENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETGGYHYS MEEMLQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFF VRLDDLFIDGLVHVSSLDNDYYRFDQVGQRLMGESSGQTYRLGDRVEVRVEAVNM DERKIDFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFR GEKKTKPKAAKKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE*(SEQ ID NO:24)
A246E MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGL RRRLRAMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYL SSEQMKTCIHGDQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFV VPDDSRLSFDILIPPDQIMGARMGFVVVVELTQRPTRRTKAVGKIVEVLGDNMGT GMAVDIALRTHEIPYIWPQAVEQQVEGLKEEVPEEAKAGRVDLRDLPLVTIDGED ARDFDDAVYCEKKRGGGWRLWVAIADVSYYVRPSTPLDREARNRGTSVYFPSQVI PMLPEVLSNGLCSLNPQVDRLCMVCEMTVSSKGRLTGYKFYEAVMSSHARLTYTK VWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKAREERGGISFESEEAKFIFNA ERRIERIEQTQRNDAHKLIEECMILANISAARFVEKAKEPALFRIHDKPSTEAIT SFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLLRSMKQAIYDP ENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETGGYHYS MEEMLQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFF VRLDDLFIDGLVHVSSLDNDYYRFDQVGQRLMGESSGQTYRLGDRVEVRVEAVNM DERKIDFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFR GEKKTKPKAAKKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE*(SEQ ID NO:25)
A246F MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGL RRRLRAMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYL SSEQMKTCIHGDQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFV VPDDSRLSFDILIPPDQIMGARMGFVVVVELTQRPTRRTKAVGKIVEVLGDNMGT GMAVDIALRTHEIPYIWPQAVEQQVFGLKEEVPEEAKAGRVDLRDLPLVTIDGED ARDFDDAVYCEKKRGGGWRLWVAIADVSYYVRPSTPLDREARNRGTSVYFPSQVI PMLPEVLSNGLCSLNPQVDRLCMVCEMTVSSKGRLTGYKFYEAVMSSHARLTYTK VWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKAREERGGISFESEEAKFIFNA ERRIERIEQTQRNDAHKLIEECMILANISAARFVEKAKEPALFRIHDKPSTEAIT SFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLLRSMKQAIYDP ENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETGGYHYS MEEMLQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFF VRLDDLFIDGLVHVSSLDNDYYRFDQVGQRLMGESSGQTYRLGDRVEVRVEAVNM DERKIDFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFR GEKKTKPKAAKKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE*(SEQ ID NO:26)
D281F MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGL RRRLRAMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYL SSEQMKTCIHGDQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFV VPDDSRLSFDILIPPDQIMGARMGFVVVVELTQRPTRRTKAVGKIVEVLGDNMGT GMAVDIALRTHEIPYIWPQAVEQQVAGLKEEVPEEAKAGRVDLRDLPLVTIDGED ARDFDFAVYCEKKRGGGWRLWVAIADVSYYVRPSTPLDREARNRGTSVYFPSQVI PMLPEVLSNGLCSLNPQVDRLCMVCEMTVSSKGRLTGYKFYEAVMSSHARLTYTK VWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKAREERGGISFESEEAKFIFNA ERRIERIEQTQRNDAHKLIEECMILANISAARFVEKAKEPALFRIHDKPSTEAIT SFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLLRSMKQAIYDP ENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETGGYHYS MEEMLQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFF VRLDDLFIDGLVHVSSLDNDYYRFDQVGQRLMGESSGQTYRLGDRVEVRVEAVNM DERKIDFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFR GEKKTKPKAAKKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE*(SEQ ID NO:27)
D281Y MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGL RRRLRAMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYL SSEQMKTCIHGDQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFV VPDDSRLSFDILIPPDQIMGARMGFVVVVELTQRPTRRTKAVGKIVEVLGDNMGT GMAVDIALRTHEIPYIWPQAVEQQVAGLKEEVPEEAKAGRVDLRDLPLVTIDGED ARDFDYAVYCEKKRGGGWRLWVAIADVSYYVRPSTPLDREARNRGTSVYFPSQVI PMLPEVLSNGLCSLNPQVDRLCMVCEMTVSSKGRLTGYKFYEAVMSSHARLTYTK VWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKAREERGGISFESEEAKFIFNA ERRIERIEQTQRNDAHKLIEECMILANISAARFVEKAKEPALFRIHDKPSTEAIT SFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLLRSMKQAIYDP ENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETGGYHYS MEEMLQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFF VRLDDLFIDGLVHVSSLDNDYYRFDQVGQRLMGESSGQTYRLGDRVEVRVEAVNM DERKIDFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFR GEKKTKPKAAKKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE*(SEQ ID NO:28)
K457F MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGL RRRLRAMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYL SSEQMKTCIHGDQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFV VPDDSRLSFDILIPPDQIMGARMGFVVVVELTQRPTRRTKAVGKIVEVLGDNMGT GMAVDIALRTHEIPYIWPQAVEQQVAGLKEEVPEEAKAGRVDLRDLPLVTIDGED ARDFDDAVYCEKKRGGGWRLWVAIADVSYYVRPSTPLDREARNRGTSVYFPSQVI PMLPEVLSNGLCSLNPQVDRLCMVCEMTVSSKGRLTGYKFYEAVMSSHARLTYTK VWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKAREERGGISFESEEAKFIFNA ERRIERIEQTQRNDAHFLIEECMILANISAARFVEKAKEPALFRIHDKPSTEAIT SFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLLRSMKQAIYDP ENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETGGYHYS MEEMLQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFF VRLDDLFIDGLVHVSSLDNDYYRFDQVGQRLMGESSGQTYRLGDRVEVRVEAVNM DERKIDFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFR GEKKTKPKAAKKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE*(SEQ ID NO:29)
T495E MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGL RRRLRAMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYL SSEQMKTCIHGDQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFV VPDDSRLSFDILIPPDQIMGARMGFVVVVELTQRPTRRTKAVGKIVEVLGDNMGT GMAVDIALRTHEIPYIWPQAVEQQVAGLKEEVPEEAKAGRVDLRDLPLVTIDGED ARDFDDAVYCEKKRGGGWRLWVAIADVSYYVRPSTPLDREARNRGTSVYFPSQVI PMLPEVLSNGLCSLNPQVDRLCMVCEMTVSSKGRLTGYKFYEAVMSSHARLTYTK VWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKAREERGGISFESEEAKFIFNA ERRIERIEQTQRNDAHKLIEECMILANISAARFVEKAKEPALFRIHDKPSTEAIE SFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLLRSMKQAIYDP ENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETGGYHYS MEEMLQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFF VRLDDLFIDGLVHVSSLDNDYYRFDQVGQRLMGESSGQTYRLGDRVEVRVEAVNM DERKIDFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFR GEKKTKPKAAKKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE*(SEQ ID NO:30)
L610W MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGL RRRLRAMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYL SSEQMKTCIHGDQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFV VPDDSRLSFDILIPPDQIMGARMGFVVVVELTQRPTRRTKAVGKIVEVLGDNMGT GMAVDIALRTHEIPYIWPQAVEQQVAGLKEEVPEEAKAGRVDLRDLPLVTIDGED ARDFDDAVYCEKKRGGGWRLWVAIADVSYYVRPSTPLDREARNRGTSVYFPSQVI PMLPEVLSNGLCSLNPQVDRLCMVCEMTVSSKGRLTGYKFYEAVMSSHARLTYTK VWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKAREERGGISFESEEAKFIFNA ERRIERIEQTQRNDAHKLIEECMILANISAARFVEKAKEPALFRIHDKPSTEAIT SFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLLRSMKQAIYDP ENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETGGYHYS MEEMWQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFF VRLDDLFIDGLVHVSSLDNDYYRFDQVGQRLMGESSGQTYRLGDRVEVRVEAVNM DERKIDFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFR GEKKTKPKAAKKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE*(SEQ ID NO:31)
V687N MSQDPFQEREAEKYANPIPSREFILEHLTKREKPASRDELAVELHIEGEEQLEGL RRRLRAMERDGQLVFTRRQCYALPERLDLVKGTVIGHRDGYGFLRVEGRKDDLYL SSEQMKTCIHGDQVLAQPLGADRKGRREARIVRVLVPKTSQIVGRYFTEAGVGFV VPDDSRLSFDILIPPDQIMGARMGFVVVVELTQRPTRRTKAVGKIVEVLGDNMGT GMAVDIALRTHEIPYIWPQAVEQQVAGLKEEVPEEAKAGRVDLRDLPLVTIDGED ARDFDDAVYCEKKRGGGWRLWVAIADVSYYVRPSTPLDREARNRGTSVYFPSQVI PMLPEVLSNGLCSLNPQVDRLCMVCEMTVSSKGRLTGYKFYEAVMSSHARLTYTK VWHILQGDQDLREQYAPLVKHLEELHNLYKVLDKAREERGGISFESEEAKFIFNA ERRIERIEQTQRNDAHKLIEECMILANISAARFVEKAKEPALFRIHDKPSTEAIT SFRSVLAELGLELPGGNKPEPRDYAELLESVADRPDAEMLQTMLLRSMKQAIYDP ENRGHFGLALQSYAHFTSPIRRYPDLTLHRAIKYLLAKEQGHQGNTTETGGYHYS MEEMLQLGQHCSMAERRADEATRDVADWLKCDFMLDQVGNVFKGVISSVTGFGFF VRLDDLFIDGLVHVSSLDNDYYRFDQNGQRLMGESSGQTYRLGDRVEVRVEAVNM DERKIDFSLISSERAPRNVGKTAREKAKKGDAGKKGGKRRQVGKKVNFEPDSAFR GEKKTKPKAAKKDARKAKKPSAKTQKIAAATKAKRAAKKKVAE*(SEQ ID NO:32)
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 Enzyme activity detection 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
Enzymes Enzyme activity (U/mu L) Relative enzyme Activity (%)
WT 6.67 100.00
D122L 7.12 106.75
K205L 6.12 91.75
A246E 8.32 124.74
A246F 7.34 110.04
D281F 7.36 110.34
D281Y 6.34 95.05
K457F 8.12 121.74
T495E 9.17 137.48
L610W 5.12 76.76
V687N 6.13 91.90
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. A mutant ribonuclease R is characterized by comprising a mutation based on a parent ribonuclease R, wherein the mutation is T495E, 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 T495E, and the amino acid sequence of the mutant T495E is as set forth in SEQ ID NO. 30.
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 strain cells to obtain an 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 in the step (3), and washing and cracking;
(5) Carrying out protein purification on the lysate obtained after the cleavage in the step (4);
(6) And (3) carrying out enzyme activity detection on the protein obtained after the purification in the step (5).
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 isopropyl-beta-D-thiogalactoside (IPTG) is added into a culture medium to a final concentration of 0.1-1.5 mM, and the fermentation is induced at 16-37 ℃ for 8-16 h, so that the mutant ribonuclease R can be induced and expressed.
17. The method according to claim 15 or 16, wherein step (4) comprises centrifuging the fermentation broth obtained in step (3) at 8000 rpm for 5 min and discarding the supernatant, then re-suspending the cells with 50mM PBS and centrifuging at 8000 rpm for 5 min and discarding the supernatant, adding PBS and re-suspending the cells, and mixing; sonicating cells on ice; the cell disruption solution was placed in a pre-chilled centrifuge, centrifuged at 8000 rpm for 10 min 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: the mutant ribonuclease R is subjected to serial gradient dilution, 10 mug of linear mRNA is degraded by the diluted mutant ribonuclease R under the condition of 37 ℃,1 h is detected by gel electrophoresis, and the enzyme activity of the mutant ribonuclease R is calculated according to the use amount of the enzyme without obvious bands.
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.
CN202211610204.8A 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof Active CN116240199B (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CN202311371859.9A CN117327678A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202311371640.9A CN117363596A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202311372194.3A CN117402854A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202311371681.8A CN117210438A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202211610204.8A CN116240199B (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202211610204.8A CN116240199B (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof

Related Child Applications (4)

Application Number Title Priority Date Filing Date
CN202311372194.3A Division CN117402854A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202311371681.8A Division CN117210438A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202311371859.9A Division CN117327678A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202311371640.9A Division CN117363596A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof

Publications (2)

Publication Number Publication Date
CN116240199A CN116240199A (en) 2023-06-09
CN116240199B true CN116240199B (en) 2023-11-07

Family

ID=86628583

Family Applications (5)

Application Number Title Priority Date Filing Date
CN202311371640.9A Pending CN117363596A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202211610204.8A Active CN116240199B (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202311372194.3A Pending CN117402854A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202311371859.9A Pending CN117327678A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202311371681.8A Pending CN117210438A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202311371640.9A Pending CN117363596A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof

Family Applications After (3)

Application Number Title Priority Date Filing Date
CN202311372194.3A Pending CN117402854A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202311371859.9A Pending CN117327678A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof
CN202311371681.8A Pending CN117210438A (en) 2022-12-14 2022-12-14 Mutant ribonuclease R and application thereof

Country Status (1)

Country Link
CN (5) CN117363596A (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110923217A (en) * 2019-12-11 2020-03-27 南京大学 Ribonuclease R capable of recognizing 2' -O-methylation modified RNA and application thereof
CN114438054A (en) * 2022-01-28 2022-05-06 广州吉赛生物科技股份有限公司 Mutant RNase R and preparation method and application thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110923217A (en) * 2019-12-11 2020-03-27 南京大学 Ribonuclease R capable of recognizing 2' -O-methylation modified RNA and application thereof
CN114438054A (en) * 2022-01-28 2022-05-06 广州吉赛生物科技股份有限公司 Mutant RNase R and preparation method and application thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
RNase R mutants eclucidate the catalysis of structured RNA: RNA-binding domains select the RNAs targeted for degradation.;Rute Goncalves Matos, et al.;Biochem J;第423卷(第2期);291-301 *

Also Published As

Publication number Publication date
CN117327678A (en) 2024-01-02
CN117363596A (en) 2024-01-09
CN117210438A (en) 2023-12-12
CN116240199A (en) 2023-06-09
CN117402854A (en) 2024-01-16

Similar Documents

Publication Publication Date Title
CN107201352B (en) β -galactosidase combined mutant with high transglycosidic activity and preparation method and application thereof
CN115161303B (en) Phospholipase mutant and method for synthesizing glycerophospholipids by using phospholipase mutant
CN110628738B (en) Method for improving activity of glucose oxidase, mutant and application thereof
CN114262697B (en) Bsu DNA polymerase and Bsu DNA polymerase mutant as well as gene, plasmid and genetic engineering bacteria thereof
CN114107252B (en) CL7 protein, high-activity recombinant TET enzyme CL7-NgTET1, prokaryotic expression vector and application
CN111410695B (en) Chimeric molecule based on autophagy mechanism mediated Tau protein degradation and application thereof
CN116240199B (en) Mutant ribonuclease R and application thereof
CN110358751B (en) Recombinant lipase mutant, encoding gene, recombinant engineering bacterium and application
CN114774399B (en) Method for artificially modifying single-base resolution positioning analysis of 5-hydroxymethylcytosine modification in deaminase-assisted DNA
CN114573673B (en) Two-fork rhinoceros scarab epidermis protein, coding nucleotide sequence and application thereof
CN110819609B (en) Mutant lipase with improved thermal stability as well as preparation method and application thereof
CN110184289B (en) Recombinant glycerol phosphate oxidase expression vector and establishment method thereof
CN114230644A (en) GP32 protein mutant, recombinant vector, and construction method and application thereof
CN115074361A (en) Strong promoter from fungus and application thereof
CN117286192B (en) Use of amide synthetases in the preparation of litaxetil intermediates and/or litaxetil
CN114015664B (en) Luciferase mutant and application thereof
CN117165551A (en) Methionine adenosyltransferase mutant and application thereof
CN116515781A (en) Phosphoglycerate oxidase, mutant thereof, preparation method and application
CN118086243A (en) Taq DNA polymerase mutant and application thereof
CN116926034A (en) Recombinant creatine kinase isozyme, and preparation method and application thereof
KR102043363B1 (en) Pectin degrading enzymes from Macrophomina phaseolina and uses thereof
JP4168130B2 (en) Polygalacturonase and polygalacturonase genes of Rhizopus sp.
JP4555920B2 (en) Protein mass expression system by mRNA stability control
CN116497001A (en) Taq DNA polymerase mutant
KR20230058876A (en) Variant of 7 beta-hydroxysteroid dehydrogenase from Ruminococcus gnavus for the conversion rate increasement of 7-Keto-LCA to UDCA and a method for producing UDCA using the same

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant