CN115927246B - Vaccinia capping enzyme mutant with high capping efficiency - Google Patents
Vaccinia capping enzyme mutant with high capping efficiency Download PDFInfo
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Abstract
The invention provides a vaccinia capping enzyme mutant with high capping efficiency. Through protein structure modeling analysis, mutant expression screening, the 529-543 disordered sequence in the D1 subunit is replaced, 3 amino acid mutations are introduced into the MTase structural domain, his tag is fused at the C end of the small subunit D12, and the high-purity mutant capping enzyme is prepared and obtained, and mRNA capping efficiency is evaluated by an LC-MS mass spectrometry method, wherein the mutant has 14.4% higher capping efficiency than that of the wild type capping enzyme. The KCE-002 mutant capping enzyme is suitable for the production of GMP-grade raw material enzyme for industrial application.
Description
Technical Field
The invention relates to the field of enzyme engineering, in particular to a vaccinia capping enzyme mutant with high capping efficiency.
Background
The vaccinia virus capping enzyme (Vaccinia Capping Enzyme) can catalyze the in vitro transcription of synthesized mRNA and add a Cap structure (Cap 0), so that the stability and translation efficiency of the mRNA are remarkably improved, and the vaccinia virus capping enzyme becomes a key raw material enzyme in the production of mRNA vaccines.
Vaccinia virus capping enzyme consists of two subunits, D1 and D12, the D1 subunit containing three domains of RNA triphosphatase (TPase), guanylase (GTase) and methyltransferase (G-N7 MTase). MTase needs to bind to D12 to function effectively. The enzymatic capping process first hydrolyzes the 5 'gamma-phosphate of mRNA by RNA triphosphatase (TPase) to produce 5' beta-phosphate. Guanylate transferase (GTase) uses GTP as substrate to form covalent enzyme-GMP intermediate, and combines beta-phosphate at mRNA 5' end with GMP to form 5' -5' connected Gppp-RNA non-methyl cap structure. Finally, the methyl transferase (G-N7 MTase) is methylated at the guanosine N7 position using ademetionine (AdoMet/SAM) as a substrate to form the Cap0 structure (m 7 GpppN) [1]. The Cap0 structure can be further modified to Cap1 (m 7 GpppmN) under the action of dioxymethyltransferase (2' O-methyltransgerase).
D1- (1-545) comprises two domains of TPase-GTase, wherein 529-543 is a disordered sequence in the crystal structure, connecting the C-terminal MTase domain D1- (540-844). IHYSF (681-685) and VLAIDFGNG (594-602) are highly conserved across multiple species. Studies show that G600 is a key amino acid affecting the catalytic function of MTase, and N550, Y555, F556, R560, R562, N570, K573, K607, Y608, D676, F679, H682 and E763 are also responsible for MTase activity.
The vaccinia virus capping enzyme D1/D12 double subunit is co-expressed in escherichia coli, his tag is fused at the N end of large subunit D1 subunit, however, D1 subunit recombinant expression is easy to degrade, after His tag affinity purification in the first step, the D1/D12 complex has more impurity bands, and finally, high-purity and high-yield capping enzyme is difficult to obtain.
Disclosure of Invention
According to the invention, through protein structure modeling analysis and mutant expression screening, a 529-543 disordered sequence in a D1 subunit is replaced by a linker sequence GGLERGRGRGRGRSRGRGSAMGGS with a structural design, 3 amino acid mutations [ Y668R ] [ F669N ] [ N753I ] are introduced in an MTase structural domain, his tag is fused at the C end of a small subunit D12, and a high-purity mutant capping enzyme (KCE-002) is prepared, mRNA capping efficiency is evaluated through an LC-MS mass spectrometry method, and the mutant has 14.4% higher capping efficiency than that of the wild type capping enzyme. The KCE-002 mutant capping enzyme is suitable for the production of GMP-grade raw material enzyme for industrial application.
In a first aspect, the invention discloses a vaccinia capping enzyme mutant comprising the D1 subunit and the D12 subunit, characterized in that: the amino acid sequence of the D1 subunit is shown in SEQ ID NO. 3, and the amino acid sequence of the D12 subunit is shown in SEQ ID NO. 5. Or a sequence having a homology of 90% or more with the amino acid sequence.
The vaccinia capping enzyme mutant is characterized in that the gene sequence of the D1 subunit is shown in SEQ ID NO. 4, and the gene sequence of the D12 subunit is shown in SEQ ID NO. 6. Or a sequence having a homology of 90% or more with the amino acid sequence.
In a second aspect, the invention discloses a recombinant expression vector, which is characterized in that: comprising the gene sequences of the D1 subunit and the D12 subunit.
Preferably, the recombinant expression vector is characterized in that: it is kana+ resistant.
Preferably, the recombinant expression vector is characterized in that: the vector is a pET-Duet recombinant plasmid vector.
In a third aspect, the present invention discloses a recombinant expression transformant characterized in that: comprising the recombinant expression vector.
Preferably, the recombinant expression transformant according to the above is characterized in that: the host cell is E.coli.
More preferably, the recombinant expression transformant according to the above is characterized in that: the host cell is Escherichia coli BL21 (DE 3) strain.
Compared with the prior art, the invention has the following technical advantages:
the KCE-002 mutant capping enzyme introduces three amino acid mutation points [ Y668R ], [ F669N ], [ N753I ] in the methylation transferase (MTase) domain of the D1 subunit; the unordered Linker sequence F529-N543 (FNEDKLSDVGHQYAN) was replaced with a GGLERGRGRGRGRSRGRGSAMGGS sequence; his tag is fused at the C-terminal of the D12 subunit.
2. The affinity tag His tag is fused at the C end of the D12 subunit, and the D1 (KCE-002)/D12 < His tag > double subunit is co-expressed in the cell of the escherichia coli BL21 (DE 3), so that the high-purity KCE-002 mutant can be prepared and obtained.
3. The KCE-002 mutant has higher capping efficiency, and the capping efficiency of the same enzyme amount is improved by 14.4% compared with that of the wild type vaccinia Capping Enzyme (CE). I.e. the same capping efficiency was obtained, the KCE-002 mutant could be reduced by 14.4% compared to the wild type.
KCE-002 mutant is suitable for industrial application in the production of GMP grade raw material enzyme.
Drawings
FIG. 1 is a diagram of a KCE-002 mutant protein electrophoresis gel of the present invention.
FIG. 2 is a deconvolution mass spectrum (Deconvoluted mass spectra) of a vaccinia capping enzyme capping mRNA nucleotide fragment of the invention.
FIG. 3 is a comparison of capping efficiencies of wild-type vaccinia Capping Enzyme (CE) of the present invention and 7 mutant capping enzymes (KCE-001-KCE-007).
Detailed Description
The following describes in further detail the embodiments of the present invention with reference to examples and drawings. The following examples and figures are illustrative of the invention and are not intended to limit the scope of the invention.
Experimental materials:
6545XT AdvanceBio LC/Q-TOF; 1290 Infinity II LC System: Agilent
LC Column AdvanceBio Oligonucleotide, 2.7 µm, 2.1 × 100 mm,120 Å: Agilent
Dynabeads MyOne Streptavidin C1: Thermo Fisher Scientific
RNAse H; Monarch RNA clean up kit: NEB
t7 RNA Polymerase (Cat. T7P-EE 101); mRNA Cap 2' -O-methyl transfer ferrose (Cat. MEH-VE 101): biotechnology (Shanghai) Co., ltd
HPLC grade Hexafluoroisopropanol (HFIP): yizhou Rundong chemical Co.Ltd
HPLC grade methanol; triethylamine (TEA): shanghai An Spectrum experiment science and technology Co.Ltd
High-pressure homogenizer: shanghai excitation ultra-high pressure equipment Co.Ltd
AKTA protein purifier: GE healthcare
Nickel-charged IMAC resin: GE healthcare
Q-HP, SP-HP pre-packed column: GE healthcare
Sephacryl S-200 molecular sieve column: GE healthcare
Protein A280 concentration determination Nanodrop Thermo Fisher Scientific
Imidazole (imidozole): sigma-Aldrich
Tris-base, naCl, tritonX100 and other chemical reagents: group of Chinese medicine
SDS-page gel electrophoresis system: tanon Shanghai energy technology Co.Ltd
Amicon Ultra-15 Centrifugal Filters Ultracel-30K: Merck
Example 1 E.coli codon optimized Gene Synthesis and construction of D1/D12 Co-expression vector
Wild type D1, D12 E.coli codon optimized genes were synthesized by general biosystems (Anhui) Inc., constructed into pET-Duet (Amp+) vector with NcoI/HindIII, ndeI/XhoI and sequenced correctly. the expression vector was engineered by Shanghai Biotechnology Inc., amp+ resistance was engineered to Kana+ resistance and sequencing was correct. On the basis, a mutation sequence is further introduced into the D1 subunit, so that the KCE-002 mutant expression vector with correct sequencing is obtained, and the KCE-002-pET-Duet (Kana+) expression vector is obtained.
Vaccinia virus capping enzyme wild-type (CE) and mutant KCE-002 sequences
Uniprot P04298 vaccinia capping enzyme wild-type (CE) D1 subunit (SEQ ID NO: 1) MDANVVSSST IATYIDALAK NASELEQRST AYEINNELEL VFIKPPLITL TNVVNISTIQ ESFIRFTVTN KEGVKIRTKI PLSKVHGLDV KNVQLVDAID NIVWEKKSLV TENRLHKECL LRLSTEERHI FLDYKKYGSS IRLELVNLIQ AKTKNFTIDF KLKYFLGSGA QSKSSLLHAI NHPKSRPNTS LEIEFTPRDN ETVPYDELIK ELTTLSRHIF MASPENVILS PPINAPIKTF MLPKQDIVGL DLENLYAVTK TDGIPITIRV TSNGLYCYFT HLGYIIRYPV KRIIDSEVVV FGEAVKDKNW TVYLIKLIEP VNAINDRLEE SKYVESKLVD ICDRIVFKSK KYEGPFTTTS EVVDMLSTYL PKQPEGVILF YSKGPKSNID FKIKKENTID QTANVVFRYM SSEPIIFGES SIFVEYKKFS NDKGFPKEYG SGKIVLYNGV NYLNNIYCLE YINTHNEVGI KSVVVPIKFI AEFLVNGEIL KPRIDKTMKY INSEDYYGNQ HNIIVEHLRD QSIKIGDIFN EDKLSDVGHQ YANNDKFRLN PEVSYFTNKR TRGPLGILSN YVKTLLISMY CSKTFLDDSN KRKVLAIDFG NGADLEKYFY GEIALLVATD PDADAIARGN ERYNKLNSGI KTKYYKFDYI QETIRSDTFV SSVREVFYFG KFNIIDWQFA IHYSFHPRHY ATVMNNLSEL TASGGKVLIT TMDGDKLSKL TDKKTFIIHK NLPSSENYMS VEKIADDRIV VYNPSTMSTP MTEYIIKKND IVRVFNEYGF VLVDNVDFAT IIERSKKFIN GASTMEDRPS TRNFFELNRG AIKCEGLDVE DLLSYYVVYV FSKR
Uniprot P20980 vaccinia capping enzyme wild type (CE) subunit D12 (SEQ ID NO: 2) MDEIVKNIRE GTHVLLPFYE TLPELNLSLG KSPLPSLEYG ANYFLQISRV NDLNRMPTDM LKLFTHDIML PESDLDKVYE ILKINSVKYY GRSTKADAVV ADLSARNKLF KRERDAIKSN NHLTENNLYI SDYKMLTFDV FRPLFDFVNE KYCIIKLPTL FGRGVIDTMR IYCSLFKNVR LLKCVSDSWL KDSAIMVASD VCKKNLDLFM SHVKSVTKSS SWKDVNSVQF SILNDPVDTE FINKFLEFSN RVYEALYYVH SLLYSSMTSD SKSIENKHQR RLVKLLL
D1 Mutant amino acid sequence [ Y668R ] [ F669N ] [ N753I ] (KCE-002) (SEQ ID NO: 3)
MDANVVSSSTIATYIDALAKNASELEQRSTAYEINNELELVFIKPPLITLTNVVNISTIQESFIRFTVTNKEGVKIRTKIPLSKVHGLDVKNVQLVDAIDNIVWEKKSLVTENRLHKECLLRLSTEERHIFLDYKKYGSSIRLELVNLIQAKTKNFTIDFKLKYFLGSGAQSKSSLLHAINHPKSRPNTSLEIEFTPRDNETVPYDELIKELTTLSRHIFMASPENVILSPPINAPIKTFMLPKQDIVGLDLENLYAVTKTDGIPITIRVTSNGLYCYFTHLGYIIRYPVKRIIDSEVVVFGEAVKDKNWTVYLIKLIEPVNAINDRLEESKYVESKLVDICDRIVFKSKKYEGPFTTTSEVVDMLSTYLPKQPEGVILFYSKGPKSNIDFKIKKENTIDQTANVVFRYMSSEPIIFGESSIFVEYKKFSNDKGFPKEYGSGKIVLYNGVNYLNNIYCLEYINTHNEVGIKSVVVPIKFIAEFLVNGEILKPRIDKTMKYINSEDYYGNQHNIIVEHLRDQSIKIGDIGGLERGRGRGRGRSRGRGSAMGGSNDKFRLNPEVSYFTNKRTRGPLGILSNYVKTLLISMYCSKTFLDDSNKRKVLAIDFGNGADLEKYFYGEIALLVATDPDADAIARGNERYNKLNSGIKTKYYKFDYIQETIRSDTFVSSVREVFrnGKFNIIDWQFAIHYSFHPRHYATVMNNLSELTASGGKVLITTMDGDKLSKLTDKKTFIIHKNLPSSENYMSVEKIADDRIVVYIPSTMSTPMTEYIIKKNDIVRVFNEYGFVLVDNVDFATIIERSKKFINGASTMEDRPSTRNFFELNRGAIKCEGLDVEDLLSYYVVYVFSKR
D1 Mutant Gene sequence (KCE-002) (SEQ ID NO: 4)
ATGGATGCAAATGTTGTTAGCAGCAGCACCATTGCCACCTATATTGATGCCCTGGCAAAGAATGCCAGTGAACTGGAACAGCGTAGCACCGCATACGAAATTAATAATGAACTGGAACTGGTGTTTATCAAACCGCCGCTGATTACCCTGACCAATGTGGTGAATATTAGTACCATTCAGGAAAGTTTCATCCGTTTCACCGTGACCAATAAAGAAGGTGTTAAGATTCGTACCAAGATTCCGCTGAGTAAAGTTCATGGCCTGGATGTTAAGAATGTGCAGCTGGTGGATGCAATTGATAATATTGTGTGGGAGAAGAAATCACTGGTGACCGAGAATCGTCTGCATAAAGAATGCCTGCTGCGTCTGAGTACCGAAGAACGCCATATCTTTCTGGATTATAAGAAATATGGCAGCAGCATTCGTCTGGAACTGGTTAATCTGATTCAGGCAAAGACCAAGAATTTCACCATTGATTTCAAACTGAAGTACTTTCTGGGTAGTGGTGCCCAGAGCAAATCAAGCCTGCTGCATGCCATTAATCATCCGAAATCTCGCCCGAATACCAGTCTGGAAATTGAATTTACACCTCGTGATAATGAAACCGTGCCGTATGATGAACTGATTAAAGAACTGACCACCCTGAGCCGCCATATCTTTATGGCAAGCCCGGAGAATGTTATTCTGAGCCCGCCGATTAATGCACCGATTAAGACTTTCATGCTGCCGAAACAGGATATTGTGGGCCTGGATCTGGAGAATCTGTACGCCGTGACCAAGACTGATGGCATTCCGATTACCATTCGTGTGACCAGCAATGGCCTGTATTGTTATTTCACCCATCTGGGTTATATTATTCGTTATCCGGTTAAACGCATTATTGATAGCGAAGTTGTGGTGTTTGGTGAAGCAGTGAAAGATAAGAATTGGACCGTTTATCTGATTAAACTGATTGAACCAGTTAATGCCATTAATGATCGCCTGGAAGAAAGCAAATATGTTGAAAGTAAACTGGTGGATATCTGCGATCGCATTGTGTTTAAATCAAAGAAATACGAAGGCCCGTTTACCACCACCAGCGAAGTGGTTGATATGCTGAGCACCTATCTGCCGAAACAACCGGAAGGTGTGATTCTGTTCTATAGTAAAGGCCCGAAATCTAATATTGATTTCAAGATTAAGAAGGAGAACACCATTGATCAGACCGCCAATGTGGTGTTTCGCTATATGAGTAGTGAACCGATTATCTTTGGCGAAAGTAGTATCTTTGTGGAATATAAGAAATTCAGCAACGATAAAGGTTTCCCGAAAGAATATGGCAGCGGTAAGATTGTGCTGTATAATGGCGTTAATTATCTGAATAACATCTACTGCCTGGAATATATTAACACCCATAATGAAGTGGGCATTAAATCTGTGGTTGTTCCGATTAAATTCATTGCAGAATTTCTGGTGAACGGCGAAATTCTGAAACCGCGCATTGATAAGACTATGAAATATATTAACAGCGAGGATTACTACGGTAATCAGCATAATATTATCGTTGAACATCTGCGCGATCAGAGTATTAAGATTGGCGATATCGGTGGTCTCGAGAGGGGACGGGGGCGAGGTAGAGGACGTTCACGCGGTCGTGGCAGCGCCATGGGCggcagtAATGATAAATTCCGTCTGAACCCGGAAGTTAGTTATTTCACCAATAAACGCACCCGTGGTCCGCTGGGTATTCTGAGTAATTATGTGAAGACTCTGCTGATTAGTATGTATTGTAGTAAGACTTTCCTGGATGATAGTAATAAACGCAAAGTGCTGGCCATTGATTTCGGTAATGGCGCCGATCTGGAGAAATATTTCTATGGCGAAATTGCACTGCTGGTGGCAACCGATCCGGATGCCGATGCAATTGCCCGTGGTAATGAACGCTATAATAAACTGAATAGCGGTATTAAGACTAAGTATTATAAGTTCGACTACATCCAGGAAACCATTCGTAGTGATACCTTTGTTAGTAGTGTTCGTGAAGTGTTCcgcaatGGCAAATTTAATATCATCGACTGGCAGTTTGCAATTCATTATAGCTTTCATCCGCGTCATTATGCCACCGTTATGAATAATCTGAGTGAACTGACCGCCAGTGGTGGCAAAGTGCTGATTACCACAATGGATGGCGATAAACTGTCAAAGCTGACCGATAAGAAGACTTTCATTATTCATAAGAACCTGCCGAGTAGCGAGAATTATATGAGTGTTGAGAAGATTGCAGATGATCGTATTGTGGTTTATATTCCGAGTACCATGAGTACACCTATGACCGAATATATTATTAAGAAGAACGACATCGTGCGTGTGTTTAATGAATATGGTTTCGTGCTGGTGGATAATGTTGATTTCGCAACCATTATTGAACGTAGTAAGAAATTTATCAACGGCGCAAGCACAATGGAAGATCGTCCGAGTACCCGCAATTTCTTTGAACTGAATCGCGGCGCCATTAAATGCGAAGGTCTGGATGTGGAAGATCTGCTGAGCTATTATGTTGTGTATGTGTTTAGCAAACGCTAA
D12< His tag > amino acid sequence (KCE-002) (SEQ ID NO: 5)
MDEIVKNIREGTHVLLPFYETLPELNLSLGKSPLPSLEYGANYFLQISRVNDLNRMPTDMLKLFTHDIMLPESDLDKVYEILKINSVKYYGRSTKADAVVADLSARNKLFKRERDAIKSNNHLTENNLYISDYKMLTFDVFRPLFDFVNEKYCIIKLPTLFGRGVIDTMRIYCSLFKNVRLLKCVSDSWLKDSAIMVASDVCKKNLDLFMSHVKSVTKSSSWKDVNSVQFSILNNPVDTEFINKFLEFSNRVYEALYYVHSLLYSSMTSDSKSIENKHQRRLVKLLLggsHHHHHH
D12< His tag > Gene sequence (KCE-002) (SEQ ID NO: 6)
ATGGATGAAATCGTGAAGAATATCCGCGAAGGTACCCATGTGCTGCTGCCGTTCTATGAAACCCTGCCGGAACTGAATCTGAGCCTGGGTAAATCTCCGCTGCCGAGTCTGGAATATGGTGCAAATTATTTCCTGCAGATTAGTCGTGTGAATGATCTGAATCGCATGCCGACCGATATGCTGAAACTGTTTACCCATGATATTATGCTGCCGGAAAGCGATCTGGATAAAGTTTATGAAATTCTGAAGATCAACAGCGTTAAATATTACGGCCGTAGCACCAAAGCCGATGCAGTGGTGGCAGATCTGAGTGCCCGCAATAAACTGTTTAAACGTGAACGTGATGCAATTAAATCTAATAATCACCTGACCGAGAATAATCTGTATATTAGCGATTATAAGATGCTGACCTTTGATGTGTTTCGCCCGCTGTTTGATTTCGTGAATGAGAAATATTGTATCATCAAGCTGCCGACCCTGTTTGGCCGCGGCGTTATTGATACCATGCGTATTTATTGTAGTCTGTTTAAGAATGTGCGTCTGCTGAAATGTGTTAGCGATAGCTGGCTGAAAGATAGCGCAATTATGGTTGCAAGCGATGTGTGCAAGAAGAATCTGGATCTGTTTATGAGTCATGTGAAATCTGTGACCAAATCAAGTAGTTGGAAAGATGTTAATAGCGTGCAGTTTAGTATTCTGAATAATCCGGTGGATACCGAATTTATTAATAAATTTCTGGAGTTCAGCAACCGCGTTTATGAAGCCCTGTATTATGTGCATAGTCTGCTGTATAGCAGCATGACCAGCGATAGCAAATCAATTGAGAATAAACATCAGCGCCGCCTGGTGAAACTGCTGCTGggtggatctCATCATCACCATCATCATtaa
EXAMPLE 2 preparation of vaccinia capping enzyme mutant
1) The vaccinia capping enzyme mutant escherichia coli expression plasmid is transformed into BL21 (DE 3) expression strain, and the strain is induced for 16 hours at a low temperature of 20 ℃ and 0.2mM IPTG, and then the thallus is obtained.
2) Buffer a (50 mm tris, ph8.0, 500mM NaCl, 10%Glycerol,0.1mM EDTA,1mM DTT,0.1% triton x 100) was used with RNase free according to cell mass (g) and buffer a volume (ml) (1: 8) The thalli are resuspended, crushed by a high-pressure refiner, and the cracked supernatant is collected by centrifugation.
3) Ni column (Nickel-charged IMAC) affinity enrichment, equilibrated column with buffer A, lysates supernatant loading hanging column, column wash with buffer A containing 10mM imidozole, gradient elution of protein with buffer A containing 500mM imidozole, and dialysis into RNase free buffer B (50 mM Tris, pH8.0, 100mM NaCl, 10% Glycerol,0.1mM EDTA,1mM DTT,0.1% Triton-X100).
4) The Q-HP column and the SP-HP column are connected in series and balanced by buffer B, the purified dialysis protein of the Ni column in the previous step is loaded, and the Q-HP column and the SP-HP column are separated and eluted by the gradient of buffer B containing 150mM-1M NaCl. The protein of interest was eluted from the SP-HP column at a salt concentration of 250mM, 300 mM. The protein was concentrated by Amicon Ultra-15 Centrifugal Filters Ultracel-30K.
5) Further purifying the protein under the conditions of Sephacryl S-200 molecular sieve and buffer B to obtain KCE-002 vaccinia capping enzyme mutant protein shown in figure 1.
The other mutant proteins and the wild-type proteins were prepared in the same manner.
Compared with wild type vaccinia capping enzyme, KCE-002 vaccinia capping enzyme mutant protein has 20% higher yield per unit cell amount of purification preparation.
Example 3 LC-MS method for determining vaccinia capping enzyme and mutant capping efficiency thereof
(1) Capped sample handling
mRNA is prepared by in vitro transcription of T7 RNA polymerase, cap0 is obtained by in vitro catalytic capping modification of vaccinia capping enzyme, and Cap1 capping product is obtained by catalytic modification of dioxymethyl transferase (2' O-methyl transferase). LC-MS assay evaluates capping efficiency of enzymes, requiring sample processing of capped mRNA to obtain 5' end capped nucleotide fragments, for analysis and quantification. The sample treatment method comprises the following steps: the biotin-labeled probe primer is designed according to the mRNA 5'-UTR sequence, the probe primer and the mRNA are annealed and combined, the biotin label on the probe is combined on a strepitavidin C1 magnetic bead, RNAse H is digested, and a nucleotide fragment containing capping modification at the 5' -end of the mRNA is excised and eluted, and the length is usually 20-30 nucleotides. The 5 '-monophosphorylate, 5' -biphosphate, G-Cap are all uncapped byproducts, cap0, cap1 and sodium (Na) and potassium (K) ion-bound Cap1 are capping products listed in Table 1. The sample processing method is described in Label-free analysis of mRNA capping efficiency using RNase H probes and LC-MS.analytical and Bioanalytical chemistry 2016, 408:5021-5030.
TABLE 1 theory and Mass Spectrometry of nucleotide fragments of the 5' -end cleavage products of capped mRNAs
(2) LC-MS instrument analysis quantification
LC-MS apparatus analysis of the quantitative cap mRNA 5 'end cleavage product nucleotide fragments method, see Rapid Analysis of mRNA 5' Capping with High Resolution LC/MS. Agilent Application notes for details: agilent application briefs (Agilent Application note). Mass spectrometry (6545 XT advanced bio LC/Q-TOF) was used in combination with a liquid phase (1290 Infinity II LC System) using AdvanceBio Oligonucleotide, 2.7 μm, 2.1×100mm, 120 a chromatographic column with a mobile phase of Triethylamine (TEA) in solution with Hexafluoroisopropanol (HFIP). Triethylamine can be used to replace butanediamine in the application briefs. The mass spectrum deconvolution spectrum of the nucleotide fragment of the enzyme-cleaved product at the 5' end of the capped mRNA is shown in figure 2, the peak area value of each uncapped product and the peak area value of the capped product are obtained by matching the molecular weight of the corresponding nucleotide fragment and deconvolution peaks on the spectrum, and the percentage of the capping efficiency is obtained by calculating the peak area of the capped product.
Example 4 capping efficiency of wild type vaccinia Capping Enzyme (CE) and 7 mutant capping enzymes (KCE-001-KCE-007)
7 mutant capping enzymes (KCE-001-KCE-007) contain different Linker sequences (replaced 529-543 disordered sequences) or contain different amino acid mutation sites, and the mutant capping enzymes and the wild type capping enzymes are prepared by adopting the same process. Capping mRNA obtained by in vitro transcription of the same batch by taking the same enzyme amount, and analyzing capping efficiency by adopting the same sample treatment and LC-MS instrument condition. Assuming a capping efficiency of 100 for the wild-type vaccinia Capping Enzyme (CE), fig. 3 shows the ratio of capping efficiency for the different mutants to the wild-type capping enzyme, with the KCE-002 mutant capping efficiency being 14.4% higher than for the wild-type capping enzyme. Higher capping efficiency is also equivalent to lower capping enzyme costs.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (7)
1. A vaccinia virus capping enzyme mutant consisting of the D1 subunit and the D12 subunit, characterized in that: the amino acid sequence of the D1 subunit is shown as SEQ ID NO. 3, and the amino acid sequence of the D12 subunit is shown as SEQ ID NO. 5.
2. The gene encoding the mutant vaccinia virus capping enzyme of claim 1, wherein the gene sequence of the D1 subunit is shown in SEQ ID No. 4 and the gene sequence of the D12 subunit is shown in SEQ ID No. 6.
3. A recombinant expression vector, characterized in that: a gene sequence comprising the D1 subunit and the D12 subunit of claim 2.
4. A recombinant expression vector according to claim 3, wherein: it is kana+ resistant.
5. A recombinant expression vector according to claim 3, wherein: the vector is a pET-Duet recombinant plasmid vector.
6. A recombinant expression transformant characterized in that: comprising the recombinant expression vector of any one of claims 3-5; the host cell is E.coli.
7. The recombinant expression transformant according to claim 6, wherein: the host cell is Escherichia coli BL21 (DE 3) strain.
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| CN111164207A (en) * | 2017-07-27 | 2020-05-15 | 优卡瑞斯 | Novel chimeric enzyme and use thereof |
| CN111893128A (en) * | 2020-06-24 | 2020-11-06 | 苏州市泽悦生物技术有限公司 | Method for preparing recombinant eukaryotic mRNA by using prokaryotic transcription system and application thereof |
| CN114395613A (en) * | 2022-03-25 | 2022-04-26 | 苏州近岸蛋白质科技股份有限公司 | Method for detecting activity of capping enzyme of vaccinia virus |
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