CN106520796A - Recombinant N-PNGase F and encoding gene and application thereof - Google Patents
Recombinant N-PNGase F and encoding gene and application thereof Download PDFInfo
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Abstract
The invention provides a recombinant N-PNGase F and an encoding gene, a preparation method and application. Compared with codon optimization, the invention has the advantages that the yield of PNGase F obtained through recombination and expression of N-PNGase F encoding gene after codon optimization is improved by four times, and the activity is improved by 50 times. In addition, a great amount of screening and optimization is conducted to multiple ingredients in the recombinant N-PNGase F reaction buffering solution by people, so as to obtain the composition and formulation of the buffering solution capable of giving full play to the activity of the PNGase F, so that the activity of the PNGase F is superior to that of the similar products on the current market, protein structure analysis is more easily conducted to protein subjected to enzymolysis of the PNGase F, and the like.
Description
Technical field
The invention belongs to bioengineering gene field, is related to a kind of restructuring PNGase F (PNGase F) and its coding
Gene and expression and purification methods and applications.
Background technology
PNGase F (systematic naming methods:Peptide-N(N-acetyl-β-glucosaminyl)
asparagineamidaseF,EC3.5.1.52；Commonly referred to as PNGaseForglycoamidase, hereinafter referred to as PNGase F) it is main
Gram-negative bacteria to be present in and some are Eukaryotic intracellular, acyl that can be between narrow spectrum fracture sugar chain and albumen
Amine key, generates a complete sugar chain and protein, and the asparagine on protein is converted into aspartic acid.Come from brain
Contain 1062 bases, coding one in the reading frame of (F.meningosepticum) PNGase F genes of film inflammation purulence bacillus
The protein of individual 354 amino acid, 40 amino acid above are speculated as signal peptide, and remaining 314 amino acid is a molecule
Measure the albumen for 34.779kD.As PNGase F can completely cut the oligonucleotide chain of N connections from above glycopeptide and glycoprotein, will
Albumen and sugar chain are completely separable, and this is significant for the analysis for carrying out cell surface sugar chain structure and function.And
PNGase F have effect to the glycoprotein that all types of N connect, and can be converted into the asparagine residue of protein
Aspartic acid, so as to carry out de-glycosylation to glycoprotein, these characteristics make it wide variety of work in glycobiology research
Tool enzyme.
PNGase F are initially extracted from meningitis purulence bacillus and are obtained, but this is very cost and a time-consuming job,
Complex steps, low yield, every liter of zymotic fluid can only obtain 0.1～0.5mg, and meningitis purulence bacillus is not suitable for greatly for pathogen
Scale fermentation.Therefore, the PNGase F prices of commercialization are very expensive, hinder extensive application and the glycoprotein of PNGase F
Function research.Researchers are weighed to PNGase F using technique for gene engineering to solve this difficult problem
Group expression.Most representational is table that Su Yishan etc. is carried out in escherichia expression system BL21 (DE3) plasmid for 2005
Reach, the PNGase F proteins that obtain activity is 200000U/mg, but be inclusion body with LB culture medium expression-forms, refolding strategy condition
It is complicated, it is difficult to control, yield is relatively low, only 10mg/L；Although being capable of achieving part soluble-expression, but expression with M9 culture mediums
Not high, the cycle is longer, and yield is not also high.(the Highly efficient production of peptides in 2014 such as Hua：
N-glycosidase F for N-glycomics analysis, Protein Expression and Purification
97 (2014) 17-22) PNGase F are expressed in yeast expression system, and the fermentation of 5L culture mediums has been carried out, yield is 800mg/
L, activity are 172172U/mg, but PNGase F genes are not optimized in pichia yeast expression system, certain
Expression of the PNGase F in Pichia pastoris and final activity are have impact in degree.
The content of the invention
In order to overcome disadvantage mentioned above, inventor that PNGase F genes are optimized in pichia yeast expression system,
PNGase F after the codon optimization for finally giving, and by PNGase F channel genes before and after optimization to Pichia pastoris Host Strains
In strain, inventor surprisingly has found, as PNGase F have higher expression after optimization, causes PNGase engineering bacterias of recombinating
Strain is when carrying out secreting, expressing, due to glycosylation modified degree it is inconsistent so that after optimization before PNGase F activity and optimization
Compare and improve nearly 50 times, 4 times of output increased.
It is an object of the present invention to provide a kind of DNA sequence dna of coding PNGase F proteins, its base sequence such as SEQ
ID NO:Shown in 1.The sequence has carried out codon optimization for pichia yeast expression system, and which is more conducive to PNGase F complete red
Express in yeast.
It is a further object to provide a kind of PNGase F proteins, its amino acid sequence such as SEQ ID NO:3 institutes
Show.
It is a further object to provide a kind of carrier containing PNGase F genes after above-mentioned code optimization, preferably
, described carrier is pAO815, pPIC9, pPIC9K, pPIC3.5, pPIC3.5K, pPICZ α A, B, C or pGAPZ α A, B, C,
More preferably pPIC3.5K, pPICZ α A or pGAPZ α A.
It is a further object to provide a kind of Pichi strain comprising carrier described above, it is preferred that institute
Pichi strain is stated for SMD1168, GS115, KM71, X-33 or KM71H, more preferably KM71 or X-33 bacterial strains.
It is a further object to provide a kind of expression of PNGase F proteins, methods described includes following steps
Suddenly：
A. build the carrier containing above-mentioned coding PNGase F genes；
B. will proceed in Pichi strain after the vector linearization of step A, and cultivate under suitable conditions；
C. recovery purifying protein.
Carrier described above is preferably pPIC3.5K, pPICZ α A or pGAPZ α A.
Pichi strain described above is preferably KM71 or X33 bacterial strains.
It is furthermore preferred that carrier described above is pPICZ α A, and Pichi strain described above is X33 bacterial strains.
It is a further object to provide a kind of restructuring PNGase F protein purification process, the purification process is such as
Under：
A. PNGase F zymotic fluid low-temperature and high-speeds are collected by centrifugation into supernatant, add imidazoles, sodium chloride, sodium dihydrogen phosphate make
In its supernatant, final concentration is respectively 20mM, 300mM, 20mM, membrane filtration.
B. equilibration buffer pillar is used first, then pretreatment in step A is obtained with purification system
PNGase F zymotic fluids pass through prepacked column or separate filler, then with elution buffer linear elution, collect eluting peak, i.e.,
Obtain the PNGase F proteins of purifying.The level pad contains 20mM sodium dihydrogen phosphates, 300mM sodium chloride, 20mM miaows
Azoles, pH7.5；The elution buffer contains 20mM sodium dihydrogen phosphates, 300mM sodium chloride, 500mM imidazoles, pH7.5.
Above-mentioned purifying process is affinity chromatography method, and prepacked column and filler are preferably Histrap crude 5ml FF.
A kind of reaction buffer formula that can give full play to PNGaseF activity is another object of the present invention is to provide,
Preferably, including following components：SDS, NP-40, β-ME, phosphate, DTT, EDTA.
Preferably, in above-mentioned reaction buffer each component concentration：SDS (v/v) 0.1%-1%, β-ME (v/v) 0.1%-
1%, DTT (v/v) 0.1%-10%, NP-40 (v/v) 0.1-5%, phosphate 5-100mM, EDTA1-50mM, pH6.0-8.0.
Preferably, in above-mentioned reaction buffer each component concentration：SDS (v/v) 0.1%-0.5%, β-ME (v/v)
0.5%-1%, DTT (v/v) 1%-10%, NP-40 (v/v) 1-5%, phosphate 20-100mM, EDTA5-50mM, pH6.0-
8.0。
Preferably, in above-mentioned reaction buffer each component concentration：SDS (v/v) 0.5%, β-ME (v/v) 0.1%, DTT
(v/v) 1%, NP-40 (v/v) 1%, phosphate 50mM, EDTA50mM, pH8.0.
As the present invention is optimized to the gene order of PNGase F, therefore the albumen that property was expressed more in the past has larger
Change, greatly improve with the front phase specific activity of optimization and yield.Inventor is optimized to its reaction buffer simultaneously, is used
DOE softwares have carried out substantial amounts of screening and optimization to Multiple components in buffer solution and formula, and final optimization pass has obtained most filling
The buffer solution composition of PNGase F activity is waved in distribution so that the PNGase F activity for finally giving is better than similar product in the market
Product, with huge market value and wide application prospect.
Description of the drawings
Fig. 1 represents restructuring PNGase F gene order comparison diagrams before and after optimization.
Wherein optimize presequence corresponding for natural PNGase F gene nucleotide series；After optimization, sequence is corresponding for this
The gene nucleotide series of the restructuring PNGase F-opt of invention, i.e. sequence after codon optimization.
Fig. 2-a, 2-b are CAI index of the PNGase F genes in pichia yeast expression system before and after optimization.
Wherein, Fig. 2-a represent natural PNGase F gene nucleotide series CAI indexes Jing in pichia yeast expression system
Program is calculated as 0.72；Fig. 2-b represent the PNGase F-opt codons of the present invention after optimization in pichia yeast expression system
Middle CAI indexes Jing programs are calculated as 0.86.
Fig. 3-a, 3-b are PNGase F genes optimal codon frequency in Pichia anomala expression host before and after codon optimization
Rate distributed areas figure.
Wherein Fig. 3-a represent that PNGase F natural gene nucleotides sequences are listed in optimal codon frequency in Bichi yeast system
Distributed areas figure, as can be seen from the figure：There is percentage in the poor efficiency codon of PNGase F natural gene nucleotide sequences
Than for 11%；Fig. 3-b represent the PNGase F-opt codons of the present invention after the optimization optimum password in Bichi yeast system
Sub- frequency distribution administrative division map, the PNGase F-opt Codon sequences poor efficiency codons of the present invention after optimization appear as 0.
Fig. 4-a, 4-b contain for PNGase F genes average GC bases in pichia yeast expression system before and after codon optimization
Amount distributed areas figure.
Wherein, Fig. 4-a represent that PNGase F natural gene nucleotides sequences are listed in average GC alkali in pichia yeast expression system
Base content is：38.74%；Fig. 4-b represent the PNGase F codons of the present invention after optimization in pichia yeast expression system
Average GC base contentses are：42.60%.
Fig. 5-a, Fig. 5-b is the agarose gel electrophoresis figure of PNGase F gene PCR products before and after codon optimization.
Wherein, Fig. 5-a are the agarose gel electrophoresis figure of PNGase F gene PCR products before optimization.Swimming lane 1 is 500bp
DNA Ladder；Swimming lane 2 contains the restructuring PNGase F gene PCR products in XhoI and XbaI enzyme cutting site for two ends.
Fig. 5-b are the agarose gel electrophoresis figure of PNGase F-opt gene PCR products after codon optimization.Swimming lane 1 is
200bp DNA Ladder；Swimming lane 2 is produced for the restructuring PNGase F-opt gene PCRs that XhoI and XbaI enzyme cutting site are contained in two ends
Thing.
Fig. 6-a, 6-b are PNGase F difference expression plasmid building process figures before and after codon optimization.
Fig. 6-a are gene expression plasmid pPICZ-PNGase F building process figures before PNGase F optimizations；Fig. 6-b are optimization
PNGase F expression plasmids pPICZ-PNGase F-opt building process figures afterwards.
Fig. 7-a, 7-b are the expression identification figure containing PNGase F genes before codon optimization in host's engineering bacteria.Figure
Middle arrow indication is PNGase F proteins.
Fig. 7-a are to be expressed one week by methanol induction containing PNGase F gene host engineered strains before codon optimization
Afterwards, bacterium solution supernatant PAGE gel electrophoretogram.Wherein swimming lane 2 is pre-dyed albumen loading Marker of 10-250KD scopes；Swimming
Road 3-10 be by Zeocin screen containing each positive monoclonal host engineering of PNGase F genes before codon optimization
Strain culturing bacterium solution supernatant.
Fig. 7-b are to be expressed one week by methanol induction containing PNGase F gene host engineered strains before codon optimization
Afterwards, bacterium solution supernatant protein western blot figure.Wherein swimming lane 1 is pre-dyed albumen loading Marker of 10-250KD scopes；Swimming lane 2-9
It is to be trained containing each positive monoclonal host engineered strain of PNGase F genes before codon optimization by what Zeocin was screened
Bacteria liquid supernatant.
Fig. 8-a, 8-b are containing expression identification of the PNGase F-opt genes in host's engineering bacteria after codon optimization
Figure.In figure, arrow indication is PNGaseF albumen.
Fig. 8-a are to express one by methanol induction containing PNGase F-opt gene host engineered strains after codon optimization
Zhou Hou, bacterium solution supernatant PAGE gel electrophoretogram.
Fig. 8-b are to express one by methanol induction containing PNGase F-opt gene host engineered strains after codon optimization
Zhou Hou, bacterium solution supernatant protein western blot figure.Wherein swimming lane 1 is pre-dyed albumen loading Marker of 10-250KD scopes；Swimming lane 2
Be by Zeocin screen containing codon optimization after in PNGase F-opt gene host's engineered strain culture bacterium solutions
Clearly.
Fig. 9 is PNGase F zymotic fluid Jing AKTA before codon optimizationTMAvant150 affinitive layer purification chromatograms.
Figure 10 is PNGase F protein Jing HisTrapcrude FF 5ml prepacked columns SDS-PAGE after purification.Its
Pre-dyed albumen loading Marker of the middle swimming lane 1 for 10-250KD scopes；Swimming lane 2-15 is that respectively purifying is in charge of middle PNGase F eggs to Jing
In vain.
Figure 11 is PNGase F-opt zymotic fluid Jing AKTA after codon optimizationTMAvant150 affinitive layer purification chromatograms
Figure.
Figure 12 is PNGase F-opt albumen Jing HisTrapcrude FF 5ml prepacked columns SDS-PAGE electrophoresis after purification
Figure.Wherein swimming lane 1 is the non-pre-dyed albumen Marker of 10-100KD, and swimming lane 2 is PNGase F protein purification of samples before gene optimization,
Swimming lane 3 is PNGase F-opt protein purification samples after codon optimization, and in figure, arrow indication is PNGase F-opt albumen.
Figure 13 is that PNGaseF-opt is determined to Rnase B bottoms in the reaction buffer of the various compositions and concentration of optimization
The activity of thing.
Figure 13-a are the activity of PNGaseF-opt digestion Rnase B in the reaction buffer of optimization, and wherein swimming lane 1 is
(only substrate Rnase B, are not added with PNGase F to negative control, and the molecular weight indicated by arrow is glycosyl compared with high protein band
Change the Rnase B of modification, relatively low is, without glycosylation modified, only have molecular weight relatively low unmodified Jing after PNGase F digestions
Rnase B protein bands, the higher glycosylation modified band of molecular weight disappears), swimming lane 2 is 1 (only commercially available N- of blank
PNGase F F, not plus Rnase B substrates), swimming lane 3 is blank 2 (only PNGaseF-opt, not plus Rnase B substrates),
Swimming lane 4 (carries out digestion after result to Rnase B with commercially available PNGase F, substrate is only left Jing after digestion for positive control
The protein band of not glycosyafated modification), swimming lane 5 be the non-pre-dyed albumen Marker of 15-100KD, swimming lane 6-9 correspondence table 1 in 1-4 realities
Test numbering.
Figure 13-b are the activity of PNGaseF-opt digestion Rnase B in the reaction buffer of optimization, and wherein swimming lane 4 is
Experiment numbers 5-13 in the non-pre-dyed albumen Marker of 15-100KD, remaining swimming lane 1-10 correspondence table 1.
Figure 13-c are the activity of PNGaseF-opt digestion Rnase B in the reaction buffer of optimization, and wherein swimming lane 1 is
Experiment numbers 14-20 in the non-pre-dyed albumen Marker of 15-100KD, remaining swimming lane 2-8 correspondence table 1.
Figure 13-d are the activity of PNGaseF-opt digestion Rnase B in the reaction buffer of optimization, and wherein swimming lane 1 is
Experiment numbers 21-25 in the non-pre-dyed albumen Marker of 15-100KD, remaining swimming lane 2-6 correspondence table 1；Swimming lane 7 exists for PNGase F
The activity in reaction buffer (1%SDS, 2%DTT, 100mM phosphate, pH7.0) before optimization.
Figure 14 is enzyme assay result in PNGase F and PNGase F-opt after optimization reaction buffer.
Figure 14-a are PNGase F enzyme assay results, and wherein swimming lane 1 is positive control (commercially available PNGase F+
Rnase B substrates), swimming lane 2 is negative control (only Rnase B substrates), and swimming lane 3 is the non-pre-dyed albumen of 15-100KD
Marker, swimming lane 4-7 are respectively PNGase F, and enzyme amount is followed successively by 100ng, 50ng, 25ng, 10ng.
Figure 14-b are PNGase F-opt enzyme assay results, and wherein swimming lane 1 is blank (only PNGase F-
Opt, not plus Rnase B substrates), swimming lane 2 be the non-pre-dyed albumen Marker of 15-100KD, swimming lane 3 be negative control (only
Rnase B substrates, not plus PNGase F-opt), 4 enzyme amount of swimming lane be 20ng, 5 enzyme amount of swimming lane be 10ng, 6 enzyme amount of swimming lane be 5ng,
7 enzyme amount of swimming lane is 2ng, and 8 enzyme amount of swimming lane is 1ng.
Figure 15 is mirror before and after recombinant expressed Human kallikrein (rhKLK1) the Jing PNGase F-opt enzymolysis of Pichia pastoris
Determine result.Wherein swimming lane 1 is the recombinant expressed Human kallikrein (China Patent Publication No. of Pichia pastoris：CN105802989)
Before enzymolysis, swimming lane 2 is 10-250KD pre-dyed albumen marker, and swimming lane 3 is the recombinant expressed Human kallikrein enzyme of Pichia pastoris
Xie Hou, rhKLK1 (China Patent Publication No. of the swimming lane 4 for Bacillus coli expression：CN103710367A).
Figure 16 is to identify before and after Human kallikrein (rhKLK1) the Jing PNGase F-opt of CHO System reorganizations expression are digested
As a result.Wherein swimming lane 1 is 10-250KD pre-dyed albumen marker, and swimming lane 2 is the Human kallikrein of CHO System reorganizations expression
(China Patent Publication No.：CN103710368, before) digesting, swimming lane 3 is the Human kallikrein enzymolysis of CHO System reorganizations expression
Afterwards.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further, it should be appreciated that quote embodiment and be merely to illustrate the present invention
Rather than limit the scope of the present invention.
The restructuring PNGase F codon optimizations of embodiment 1
DNA sequence dna (GenBank accession number of the inventor according to the published PNGase F of GenBank：AF165910.1),
Such as SEQ ID No：Shown in 2, obtain after codon optimization being carried out to the gene PNGase F-opt genes of the present invention, nucleotides
Sequence such as SEQ ID No：Shown in 1, amino acid sequence such as SEQ ID No：Shown in 3.It is presented herein below to PNGase F codon optimizations
Each parameter comparison is as follows in front and back：
1. codon adaptation indexI (CAI)
From Fig. 2-a, before codon does not optimize, PNGase F original genes passwords in pichia yeast expression system
Sub- adaptation index (CAI) is 0.72.From Fig. 2-b, by codon optimization, PNGase F genes are in Pichia anomala expression system
In system, CAI indexes are 0.86.It is optimal high efficient expression shape in the expression system to be considered as the gene during generally CAI=1
State, CAI indexes are lower to show that gene expression in the host is poorer, it can be seen that after have passed through codon optimization
The gene order for obtaining can improve expression of the PNGase F genes in pichia yeast expression system.
2. optimal codon usage frequency (FOP)
From Fig. 3-a, based on yeast expression vector, before codon does not optimize, PNGase F gene orders
There is percentage for 11% in poor efficiency codon (codon of the utilization rate less than 40%).This gene being not optimized is adopted
With series connection rare codon, these codons may reduce translation efficiency, or even can dismiss translation assemblage.Can by Fig. 3-b
Know, after codon optimization, it is 0 that PNGase F genes occur the frequency of poor efficiency codon in Bichi yeast system.
3. GC base contentses (GC curve)
G/C content ideal distribution region is 30%-70%, and any peak of the appearance outside this region all can be to some extent
Affect transcription and translation efficiency.By Fig. 4-a, the GC base average contents distributed areas figure contrast of the PNGase F genes of Fig. 4-b
Understand, by showing PNGase F gene GC bases average content in Fig. 4-a for 38.74%, by show in Fig. 4-b optimize after go
Except the G/C content peak value occurred in addition to 30%-70% regions, after finally giving optimization, the GC base average contents of PNGase F are
42.60%.
Embodiment 2：PNGase
The expression plasmid of F and PNGase F-opt genes builds
PNGase F before and after codon optimization are introduced into XhoI restriction enzyme site sequences at 5 ' ends, histidine are introduced at 3 ' ends
Label (SEQ ID No：4) with XbaI enzyme cutting site sequence, and full genome synthesis is carried out, the genetic fragment that will synthesize is building up to
In pUC57 plasmids (being provided by Nanjing Jin Sirui Science and Technology Ltd.s), a kind of long-term preservation plasmid is obtained, by the plasmid before optimization
PUC57-PNGase F are designated as, the plasmid after optimization is designated as pUC57-PNGase F-opt plasmids.
With pUC57-PNGase F and pUC57-PNGase F-opt plasmids as template, enter performing PCR amplification, the primer sequence
Row are as follows：
Upstream primer (SEQ ID No：5)：
M13F:CGC CAG GGT TTT CCC AGT CAC GAC
Downstream primer (SEQ ID No：6)：
M13R:AGC GGA TAA CAA TTT CAC ACA GGA
Reaction 50 μ L of cumulative volume, wherein concentration respectively add 2.5 μ L, concentration to add for the dNTP of 10mmol/L for 10 μm of ol/L primers
1 μ L, archaeal dna polymerase used are Q5 (being purchased from New England Biolabs companies), 2U/ μ L, plus 0.5 μ L.Reaction condition is 98
DEG C 5 seconds, 55 DEG C 45 seconds, 72 DEG C 30 seconds, after 25 circulations, as a result the analysis of 1.0% agarose gel electrophoresis of product Jing shows product
Size is consistent (result is as shown in Fig. 5-a and Fig. 5-b) with expected size (1000bp).Xba I (R0145S, purchased from New are used respectively
England Biolabs companies) and Xho I (R0189S, purchased from New England Biolabs companies) double digestion after, 1% fine jade
Lipolysaccharide electrophoresis, the gene outcome for obtaining are pure with DNA gel QIAquick Gel Extraction Kit (DP214, Beijing Tiangeng biochemical technology Co., Ltd)
Change.PPICZ α A plasmids are connected to T4 ligases (M0202S, purchased from New England Biolabs), and (V173-20 is purchased from
Invitrogen companies) in, it is transformed in DH5 α competent cells (CB101, purchased from Beijing Tiangeng biochemical technology Co., Ltd),
37 DEG C of overnight incubations in the LB solid mediums containing bleomycin (being purchased from Invitrogen companies).Second day picking positive
Clone bacterium is sequenced, and compares, completely the same with expected sequence, that is, obtain the expression plasmid before and after PNGase F codon optimizations, point
PPICZ-PNGase F and pPICZ-PNGase F-opt (plasmid construction such as Fig. 6-a, shown in 6-b) are not designated as.
Embodiment 3：Structure containing restructuring PNGase F gene difference Pichia pastoris host's engineered strains
YPDS solid mediums are prepared：The Easy SelectPichia Expression Kit explanations of Invitrogen companies
Book is provided.
1. the structure containing PNGase F hosts engineered strain before and after codon optimization
Method according to Invitrogen companies Easy SelectPichia Expression Kit specifications is prepared into
Electrocompetent cells.The plasmid pPICZ-PNGase F and pPICZ-PNGase F-opt that embodiment 2 is obtained, are limited with Sac I
Property restriction endonuclease (R0156S, purchased from New England Biolabs) linearization for enzyme restriction processed, by linearized vector after ethanol precipitation,
Electricity conversion is entered in Pichia pastoris competent cell, coats YPDS solid mediums, and 30 DEG C of cultures are grown until transformant
Afterwards.
Embodiment 4：Containing PNGase F engineering strains abduction delivering and identification before and after codon optimization
BMGY culture mediums are prepared：Yeast extract 10g/L, peptone 20g/L, K2HPO43g/L, KH2PO411.8g/L,
YNB 13.4g/L, biotin 4 × 10-4G/L, glycerine 10g/L.
BMMY culture mediums are prepared：Yeast extract 10g/L, peptone 20g/L, K2HPO43g/L, KH2PO411.8g/L,
YNB13.4g/L, biotin 4 × 10-4G/L, methyl alcohol 5mL/L.
1. PNGase F hosts engineered strain methanol induction expression before codon optimization
Picking embodiment 3 obtain host's monoclonal engineering bacteria in 5mL BMGY culture mediums, in 50mL sterile centrifugation tubes
In 30 DEG C, 220rpm culture, during to OD600=1.0-2.0, take 1mL preserve bacterial classification, and will be remaining bacterium solution resuspended after be transferred to
BMMY Small Amount abduction deliverings, add methyl alcohol to final concentration of 1% every 24h.After one week, bacterium solution supernatant is collected by centrifugation, is passed through
(one resists for Anti-His, M20001, Abmart for PAGE gel electrophoresis and Western blot analysis；Two resist for Anti-
Rabbit IgG-Peroxidase antibody produced in goat, A0545-1mL, are purchased from Sigma-Aldrich),
Observation expression product band brightness, Fig. 7-a, Fig. 7-b are containing PNGase F engineering strain abduction delivering qualification figures.By scheming
7-a and Fig. 7-b understand that PNGase F proteins are expressed in engineered strain.
2. PNGase F-opt engineered strains methanol induction expression after codon optimization
Picking embodiment 3 obtain host's monoclonal engineering bacteria in 5mL BMGY culture mediums, in 50mL sterile centrifugation tubes
In 30 DEG C, 220rpm culture, during to OD600=1.0-2.0, take 1mL preserve bacterial classification, and will be remaining bacterium solution resuspended after be transferred to
BMMY Small Amount abduction deliverings, add methyl alcohol to final concentration of 1% every 24h.After one week, bacterium solution supernatant is collected by centrifugation, is passed through
PAGE gel electrophoresis and Western blot analysis, observe expression product band brightness, Fig. 8-a, Fig. 8-b be containing
PNGase F-opt engineering strain abduction delivering qualification figures.Existed from Fig. 8-a and Fig. 8-b, PNGase F-opt albumen
Notable expression has been obtained in engineered strain.
Embodiment 5：The purifying of PNGase F and PNGase F-opt recombinant proteins
The PNGase F and PNGase F-opt that this patent builds carries 6 × His-Tag, mainly using affinitive layer purification
PNGaseF and PNGase F-opt albumen.Prepackage pillar is selected to be HisTrapcrude 5ml FF, is comprised the following steps that：
1. the removal of impurities pretreatment of zymotic fluid
PNGase F or PNGase F-opt host's engineered strain zymotic fluids, 12000rpm, 15min are obtained by embodiment 4
Low-temperature centrifugation collects supernatant, adds sodium dihydrogen phosphate, sodium chloride and imidazoles so as to which final concentration is respectively 20mM in supernatant,
300mM, 20mM, 0.22 μm of membrane filtration.
2. Ni affinitive layer purifications
Operate with fully-automatic intelligent protein purification system (AKTA avant150, GE healthcare) UNICORN6.1
Software carries out purifying process optimization to the PNGase F and PNGase F-opt zymotic fluids that pretreatment is obtained, and is ultimately determined to balance
Buffer solution be 20mM sodium phosphates, 300mM sodium chloride, 20mM imidazoles, pH7.5, elution buffer be 20mM sodium phosphates, 300mM chlorine
Change sodium, 500mM imidazoles, pH7.5.Linear elution, collects eluting peak, PNGase F purifying chromatograms and SDS-PAGE Purities
See in Fig. 9 and Figure 10, Figure 10 that PNGF is three protein bands, above two bands be it is glycosylation modified after albumen, molecular weight compared with
The not glycosyafated modification of low band, yield is 5mg/L；PNGase F-opt purify chromatogram and SDS-PAGE Purity figures are shown in
Figure 11 and Figure 12.Figure 10,12 show gene optimizations after PNGase F proteins based on two higher bands of glycosylation modified degree,
After optimization, PNGase F-opt albumen is based on the relatively low band of glycosylation modified degree, and yield is apparently higher than before optimization, most
High reachable 20mg/L.Merge satisfactory collecting pipe after SDS-PAGE electrophoresis determines purity, filtration sterilization is accorded with
The PNGase F proteins for requiring are closed, next step active determination test is carried out.
Embodiment 6：PNGase F-optThe optimization of reaction buffer
It is more to PNGase F-opt enzymatic activity influence factors in reaction buffer：Such as denaturant, surfactant, reduction
Agent, detergent, buffer solution species, pH value, concentration etc.；Using DOE test design methods, above-mentioned influence factor is carried out excellent
Change, table 1 show the various formulas of the reaction buffer being optimized and concentration, and corresponding PNGase F-opt in each buffer body
The lower enzyme activity of system.With system comparative result before optimization, the protein actives of PNGase F-opt shown in Figure 13 are greatly improved, and work as change
Property agent concentration it is relatively low, during buffer system moderate concentration, enzyme activity obtains maximum lift, with buffer solution optimization before phase specific activity improve
220%.
Table 1：Each component and its concentration in DOE optimization reaction buffers
Embodiment 7：The measure of PNGaseF and PNGaseF-opt proteinase activities
1. by the PNGase F and PNGase F-opt that obtains of purifying with buffer solution (50mM NaCl, 20mM Tris-HCl,
5mM EDTA, 50%Glycerol, pH7.5) dialysis, Pierce BCA determination of protein concentration kit (Cat No:23225) survey
Determine protein concentration, PNGase F and PNGase F-opt are diluted to into 1mg/ml；
2. 1 μ L glycoprotein denaturation buffers (1%SDS, 1% β-ME), 100 DEG C of heating are added in 20 μ gRnase B
10min；
3. 2 μ L 10%NP-40,2 μ 10 × Glycobuffer2 of l (20mM phosphoric acid are added after solution is cooled to room temperature
Salt, 10mM EDTA, pH7.0), the PNGase F, 37 DEG C of accurate response 1h for adding 1 μ L to dilute；
4. 70 DEG C of reactant liquor heats 10min terminating reactions, SDS-PAGE identified activities；1h can be by 95% Rnase B bottoms
The PNGase F unit definitions of thing excision are 1 active unit, and Figure 14 show PNGase F and active with PNGase F-opt
Qualification result, it is 500000U/mg to be calculated PNGase F-opt specific activity of enzyme, and PNGase F specific activity of enzyme is 10000U/
Mg, after optimization, specific activity of enzyme improves 50 times.
Embodiment 8：Applications of the PNGaseF-opt in the protein structural analysis analysis that Bichi yeast system is expressed
Modification is glycosylated when antibody and recombinant protein are expressed in eukaryotic expression system easily, wherein N- sugar-type is modified to
The modal modified types of albumen, such medicine need first to remove glycosylation modified, conventional removal before peptide figure analysis are carried out
Glycosylation modified method is enzymatic isolation method, is removed with PNGase F glycosylation modified；In the present embodiment, inventor uses
PNGase F-opt recombinant expressed to Bichi yeast system restructuring Human kallikrein (rhKLK1) (China Patent Publication No.：
CN105802989 N glycosylation process) is carried out, concrete enzymolysis process is with reference to detailed step in embodiment 7；Jing N- shown in Figure 15 is sugared
After amidase F enzymolysis, rhKLK1 is single protein band, and molecular weight is substantially reduced than comparing before enzymolysis, with Bacillus coli expression
Not glycosyafated modification rhKLK1 (China Patent Publication No.：CN103710367A) compare molecular weight to be close to, illustrate rhKLK1
Glycoprotein be completely removed, Jing enzymolysis after kininogenase Argine Monohydrochloride coverage rate comply fully with requirement, illustrate this patent
In PNGase F enzymes can be applicable to structural analysis analysis.
Embodiment 9：Applications of the PNGaseF-opt in CHO system expression protein structural analysis
Albumen containing glycosylation modified site can occur N when expressing in CHO expression systems glycosylation modified, molecular weight
Much larger than theoretical molecular, thereby increases and it is possible to can make purpose band that disperse state is presented in electrophoresis, be unfavorable for protein molecular characterize and
Structural analysis.PNGase F enzymes in the present invention can be applicable to the analysis of glycoprotein structure and the identification of property, enzyme shown in Figure 16
Human kallikrein (the China Patent Publication No. of CHO System reorganizations expression before solution：CN103710368) molecular weight very disperse,
And apparently higher than after enzymolysis, Jing after PNGase F enzymolysis, (Chinese patent is disclosed the Human kallikrein of CHO System reorganizations expression
Number：CN103710368 be) homogeneous protein band, before molecular weight is significantly lower than enzymolysis, illustrate this albumen disperse be entirely by
Cause in glycosylation modified.Human kallikrein (the China Patent Publication No. of the CHO System reorganizations expression after enzymolysis：
CN103710368) because there is no glycosylated impact, advantageously in the egg such as development quality analysis research and peptide figure analysis research
White structural research.
SEQUENCE LISTING
<110>Jiangsu Zhonghong Biopharma Institute Inc.
<120>A kind of restructuring PNGase F and its encoding gene and application
<130>A kind of restructuring PNGase F and its encoding gene and application
<160> 6
<170> PatentIn version 3.3
<210> 1
<211> 948
<212> DNA
<213> Pichia pastoris
<400> 1
gccccagctg ataacaccgt taacatcaag acattcgata aggtcaaaaa cgccttcggt 60
gacggattgt ctcaatccgc agaaggtact tttactttcc cagccgacgt tactgcagtc 120
aagaccatta aaatgtttat caagaacgaa tgtcctaaca agacttgcga tgagtgggac 180
agatacgcta acgtttatgt taagaacaag actaccggtg aatggtacga gattggaaga 240
ttcattactc catattgggt tggtactgaa aagttgccta gaggacttga gattgatgtt 300
accgacttca aatcattgct tagtggtaac acagaattga agatctacac tgagacctgg 360
cttgctaaag gtagagagta ctccgttgat ttcgacattg tctatggaac tccagattac 420
aagtattcag ctgttgtccc tgttgtccaa tacaataagt cttccatcga cggtgttcca 480
tacggtaaag ctcatacttt ggccttgaag aagaacattc agttgcctac aaatactgaa 540
aaggcttact tgagaacaac tatctcaggt tggggacacg caaaaccata cgatgctggt 600
agtagaggat gtgctgagtg gtgctttaga acccatacaa ttgccatcaa caactctaac 660
actttccaac accagttggg tgcccttgga tgttctgcaa accctattaa caatcaatcc 720
ccaggaaatt ggaccccaga tagagctggt tggtgcccag gaatggccgt tcctactaga 780
attgacgtct tgaacaattc ccttatcgga tctacctttt cctacgaata taagttccag 840
aactggacaa acaatggtac taatggagat gcattctacg ctatttcaag tttcgttatc 900
gcaaaatcaa acacaccaat tagtgctcct gttgtcacta attaatag 948
<210> 2
<211> 948
<212> DNA
<213> Chryseobacterium meningosepticum
<400> 2
gctccggcag ataatacggt aaatattaaa acattcgaca aagtaaaaaa tgcctttggt 60
gacggattgt cccaaagtgc ggaaggaacc tttacatttc cggccgatgt aacagccgta 120
aaaacgatta agatgttcat taaaaatgaa tgtcctaata aaacttgtga tgaatgggat 180
cgttatgcca atgtttatgt aaaaaataaa acaacaggtg agtggtacga aataggacgc 240
tttattactc catattgggt gggaacggaa aaattacctc gtggactgga aattgatgtt 300
acagatttca aatctttact atccggaaat acagaactta aaatttatac ggagacatgg 360
ctggccaaag gaagagaata cagtgtagat ttcgatattg tatacgggac accggattat 420
aaatattcgg ctgtagtacc tgtagttcag tataacaaat catctattga cggagtccct 480
tatggtaaag cacatacatt ggctttgaaa aagaatatcc agttaccaac aaacacagaa 540
aaagcttatc ttagaactac tatttccgga tggggacatg ctaagccata tgatgcggga 600
agcagaggtt gtgcagaatg gtgcttcaga acacacacta tagcaataaa taattcgaat 660
actttccagc atcagctggg tgctttagga tgttcagcaa accctatcaa taatcagagt 720
ccgggaaatt ggactcccga cagagccggt tggtgcccgg gaatggcagt tccaacacgt 780
atagatgtac tgaataattc tttaataggc agtactttta gttatgaata taaattccag 840
aactggacaa ataacggaac caatggagat gctttttatg caatttccag ttttgtgatt 900
gcaaaaagta atacacctat tagtgctccg gtagttacaa actagtaa 948
<210> 3
<211> 314
<212> PRT
<213> Chryseobacterium meningosepticum
<400> 3
Ala Pro Ala Asp Asn Thr Val Asn Ile Lys Thr Phe Asp Lys Val Lys
1 5 10 15
Asn Ala Phe Gly Asp Gly Leu Ser Gln Ser Ala Glu Gly Thr Phe Thr
20 25 30
Phe Pro Ala Asp Val Thr Ala Val Lys Thr Ile Lys Met Phe Ile Lys
35 40 45
Asn Glu Cys Pro Asn Lys Thr Cys Asp Glu Trp Asp Arg Tyr Ala Asn
50 55 60
Val Tyr Val Lys Asn Lys Thr Thr Gly Glu Trp Tyr Glu Ile Gly Arg
65 70 75 80
Phe Ile Thr Pro Tyr Trp Val Gly Thr Glu Lys Leu Pro Arg Gly Leu
85 90 95
Glu Ile Asp Val Thr Asp Phe Lys Ser Leu Leu Ser Gly Asn Thr Glu
100 105 110
Leu Lys Ile Tyr Thr Glu Thr Trp Leu Ala Lys Gly Arg Glu Tyr Ser
115 120 125
Val Asp Phe Asp Ile Val Tyr Gly Thr Pro Asp Tyr Lys Tyr Ser Ala
130 135 140
Val Val Pro Val Val Gln Tyr Asn Lys Ser Ser Ile Asp Gly Val Pro
145 150 155 160
Tyr Gly Lys Ala His Thr Leu Ala Leu Lys Lys Asn Ile Gln Leu Pro
165 170 175
Thr Asn Thr Glu Lys Ala Tyr Leu Arg Thr Thr Ile Ser Gly Trp Gly
180 185 190
His Ala Lys Pro Tyr Asp Ala Gly Ser Arg Gly Cys Ala Glu Trp Cys
195 200 205
Phe Arg Thr His Thr Ile Ala Ile Asn Asn Ser Asn Thr Phe Gln His
210 215 220
Gln Leu Gly Ala Leu Gly Cys Ser Ala Asn Pro Ile Asn Asn Gln Ser
225 230 235 240
Pro Gly Asn Trp Thr Pro Asp Arg Ala Gly Trp Cys Pro Gly Met Ala
245 250 255
Val Pro Thr Arg Ile Asp Val Leu Asn Asn Ser Leu Ile Gly Ser Thr
260 265 270
Phe Ser Tyr Glu Tyr Lys Phe Gln Asn Trp Thr Asn Asn Gly Thr Asn
275 280 285
Gly Asp Ala Phe Tyr Ala Ile Ser Ser Phe Val Ile Ala Lys Ser Asn
290 295 300
Thr Pro Ile Ser Ala Pro Val Val Thr Asn
305 310
<210> 4
<211> 18
<212> DNA
<213> Pichia pastoris
<400> 4
catcaccatc accatcac 18
<210> 5
<211> 24
<212> DNA
<213> Pichia pastoris
<400> 5
cgccagggtt ttcccagtca cgac 24
<210> 6
<211> 24
<212> DNA
<213> Pichia pastoris
<400> 6
agcggataac aatttcacac agga 24
Claims (10)
1. a kind of gene of coding PNGase F proteins, its base sequence such as SEQ ID NO:Shown in 1.
2. the carrier containing the gene that PNGase F proteins are encoded described in claim 1, described carrier is pAO815, pPIC9,
PPIC9K, pPIC3.5, pPIC3.5K, pPICZ α A, B, C or pGAPZ α A, B, C.
3. comprising carrier described in claim 2 Pichi strain, the Pichi strain be SMD1168, GS115,
KM71, X-33 or KM71H.
4. a kind of expression of PNGase F proteins, methods described include following step：
A. build the carrier containing the gene that PNGase F proteins are encoded described in claim 1；
B. will proceed in Pichi strain after the vector linearization of step A, and cultivate under suitable conditions；
C. recovery purifying protein.
5. a kind of restructuring PNGase F protein purification process, the purification process are as follows：
A. PNGase F zymotic fluid low-temperature and high-speeds are collected by centrifugation into supernatant, add imidazoles, sodium chloride, sodium dihydrogen phosphate so as on
In clear, final concentration is respectively 20mM, 300mM, 20mM, membrane filtration；
B. equilibration buffer pillar is used first, and the PNGase F of acquisition will be pre-processed in step A with purification system then
Zymotic fluid passes through prepacked column or separates filler, then with elution buffer linear elution, collects eluting peak, that is, obtains pure
The PNGase F proteins of change；The level pad contains 20mM sodium dihydrogen phosphates, 300mM sodium chloride, 20mM imidazoles, pH7.5；
The elution buffer contains 20mM sodium dihydrogen phosphates, 300mM sodium chloride, 500mM imidazoles, pH7.5.
6. a kind of PNGaseF reaction buffers are filled a prescription, including following components：SDS, NP-40, β-ME, phosphate, DTT, EDTA.
7. in the reaction buffer described in claim 6 each component concentration：SDS (v/v) 0.1%-1%, β-ME (v/v)
0.1%-1%, DTT (v/v) 0.1%-10%, NP-40 (v/v) 0.1-5%, phosphate 5-100mM, EDTA1-50mM,
pH6.0-8.0。
8. in the reaction buffer described in claim 6 each component concentration：SDS (v/v) 0.1%-0.5%, β-ME (v/v)
0.5%-1%, DTT (v/v) 1%-10%, NP-40 (v/v) 1-5%, phosphate 20-100mM, EDTA5-50mM, pH6.0-
8.0。
9. in the reaction buffer described in claim 6 each component concentration：SDS (v/v) 0.5%, β-ME (v/v) 0.1%,
DTT (v/v) 1%, NP-40 (v/v) 1%, phosphate 50mM, EDTA50mM, pH8.0.
10. the PNGase F that PNGase F protein gene codes are encoded described in claim 1 are sweetened off in glycoprotein structure analysis
The application of base.
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| CN108690867A (en) * | 2017-04-11 | 2018-10-23 | 南京农业大学 | A kind of enzyme activity determination method of n-glycosylase |
| CN108715892A (en) * | 2018-06-14 | 2018-10-30 | 浙江农林大学 | A kind of rareness species DNA information acquisition methods |
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| CN104560908A (en) * | 2013-10-29 | 2015-04-29 | 天津强微特生物科技有限公司 | Method for purifying recombinant escherichia coli Taq DNA polymerase |
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