CN107083372A - The expression of the O sulfotransferases of recombinase Heparan sulfate 3 and its application in synthesis Heparan sulfate - Google Patents
The expression of the O sulfotransferases of recombinase Heparan sulfate 3 and its application in synthesis Heparan sulfate Download PDFInfo
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Abstract
Application the invention discloses the expression of the O sulfotransferases of recombinase Heparan sulfate 3 and its in synthesis Heparan sulfate.The RNA originated using Wistar rat cerebral tissues carries out reverse transcription for template, obtains the complementary DNA sequence of 3 O sulfate transferases 1, carries out codon optimization and cuts off signal peptide；Fragment after optimization is connected with pET 28a plasmids, imports and induced expression is carried out in Host Strains e. coli bl21 (DE3).Obtain after expression, carrying out purifying using nickel affinity column removes foreign protein；And renaturation is carried out to inclusion body using dialysis；By the phosphosulfate regenerative system of 3 ' AMP 5 ', carry out Enzyme activity assay to refolded protein, and applied to substrate N it is deacetylated/heparosan (NSNAH) of sulphation sulphation modification in.The present invention can effectively obtain the expression of the O sulfotransferases of recombinase Heparan sulfate 3, and be used successfully to synthesize Heparan sulfate.
Description
Technical field
The present invention relates to the expression of recombinase Heparan sulfate 3-O sulfotransferases and its in synthesis acetyl sulfate
Application in heparin.
Background technology
Heparan sulfate (HS) is the glycosaminoglycan that a class is widely present in cell surface, participates in a series of biological anti-
Should, such as blood clotting, wound healing, embryonic development.It is similar to the structure of heparin, is all the dissacharide units group by repeating
Into.Heparan sulfate is to be alternateed to increase to the non-reducing end of chain and formed by gucosamine and glucuronic acid.Then, lead to
A series of reaction is crossed to be modified, including N- is deacetylated, N- sulphations, C5 isomerization and diverse location and the sulfuric acid of degree
Change.Due to the imperfection of these reactions, the polysaccharide of synthesis also has different 26S Proteasome Structure and Functions.And HS and different albumen
It is exactly certainly in O- sulfation sites and degree on polysaccharide chain that interaction, which takes,.There are some researches show HS and its derivative exist in recent years
Participate in playing an important role in virus infection.The especially HS of 3-O sulphations can and antithrombase, HSV-1 glycoprotein D are fine
Tie up Porcine HGF etc. to combine, play specific function.
During Heparan sulfate 3-O sulfotransferases 1 (3-O- sulfate transferases -1) are Heparan sulfate biosynthesis
Key enzyme.Sulfate group can be transferred on the 3-O positions of gucosamine by it.The expression and application of 3-O- sulfate transferases -1
It is significant for external synthesis Heparan sulfate and its functional study.And it is current, turn both at home and abroad on 3-O- sulfuric acid
The report for moving the efficient heterogenous expression of enzyme -1 is also extremely limited, and correlative study does not also deploy deeply.
The content of the invention
It is an object of the invention to provide a kind of effective and feasible method, by building genetic engineering, recombinase sulfuric acid is obtained
The expression of heparan 3-O sulfotransferases, and be applied in the synthesis of Heparan sulfate.
To realize first goal of the invention, the technical solution adopted by the present invention is as follows：
The expression of recombinase Heparan sulfate 3-O sulfotransferases, it is characterised in that utilize Wistar rats
The RNA in brain tissue source carries out reverse transcription for template, obtains the complementary DNA sequence of 3-O- sulfate transferases -1, enters
Row codon optimization simultaneously cuts off signal peptide；Fragment after optimization is connected with pET-28a plasmids, imports Host Strains e. coli bl21
(DE3) induced expression is carried out in.
Specifically, have the following steps：
(1) real-time RT-PCR (RT-PCR)：According to the Rattus norvegicus 3-O- sulfate transferases -1 reported
Gene (GenBank accession number：NO.NM_053391.1 a pair of specific primers) are devised, restriction enzyme is added in primer
III two restriction enzyme sites of enzyme BamH I and Hind；It is poly- using Real time reverse transcription using Wistar rat brains total RNA as template
The amplification of synthase chain reaction (RT-PCR) technology obtains the genetic fragment of 3-O- sulfate transferases -1；By the obtained fragments of PCR and pMD-
19-T carriers are connected, and are imported in bacillus coli DH 5 alpha competence, are carried out with the screening of blue hickie with bacterium colony PCR
Checking；
(2) codon optimization：Rare codon analysis is carried out to sequence, while carrying out codon optimization；
(3) express：Gene after codon optimization is connected to pET-28a carriers, imports plasmid using hot robin and expresses
In host e. coli BL21 (DE3)；After resistance screening, induced using isopropylthiogalactoside (IPTG), make mesh
Protein expression；Ultrasonication is carried out to the cell after induction, liquid is crushed as total protein sample, centrifuging and taking supernatant and precipitation point
Zuo Wei not soluble and insoluble proteins sample, progress polyacrylamide gel electrophoresis (SDS-PAGE).
To realize second goal of the invention, the technical scheme of use is as follows：
Application of the recombinase Heparan sulfate 3-O sulfotransferases in synthesis Heparan sulfate, it is characterised in that
Obtain after expression, carrying out purifying using nickel affinity column removes foreign protein；And renaturation is carried out to inclusion body using dialysis；Pass through
3 '-AMP -5 '-phosphosulfate regenerative system, Enzyme activity assay is carried out to refolded protein, and second is taken off applied to substrate N-
In the heparosan (NSNAH) of acyl/sulphation sulphation modification.
Specifically, have the following steps：
(4) renaturation：For the inclusion body of inactive, it is necessary to it is correctly folded into soluble protein through renaturation.This
Experiment takes dialysis to carry out renaturation.Main method is by solubilization of inclusion bodies in denaturing liquid, and to load bag filter.Dialyzate
The denaturing liquid of the urea containing 6M is initially, 1.5L renaturation solutions are then added with 0.8 mL/min speed；
(5) enzyme activity determination：After renaturation terminates, 10000rpm centrifugation 10min take supernatant, as renaturation is successfully soluble
Destination protein.Utilize 3 '-AMP -5 '-phosphosulfate regenerative system progress enzyme activity determination.3-O- sulfate transferases -1
Enzyme activity is defined as the enzyme amount of the interior conversion 1nmol substrates of 1min or 1nmol sulfate groups under conditions of 37 DEG C, pH 7.0.Enzyme activity meter
Calculate formula as follows：
The meaning of each parameter in formula：Abst=5 and Abst=20 reactants when being enzymatic reaction 5min and 20min respectively
The OD of system400Value, ε0Molar extinction coefficient numerical value is 10.5 × 10-3, RT is the reaction time, and [3OST] is the concentration of enzyme, and 1 represents
The volume of reaction system, 1000 represent μm ol to nmol Conversion of measurement unit.
(6) -1 couple of NSNAH of 3-O- sulfate transferases sulphation modification：Reaction cumulative volume is 20ml, in experimental group successively
Add the fragrant sulfotransferase (AST-IV) of -5 '-phosphosulfate of 20mg 3 '-AMPs of NSNAH, 8mL, 5mL, 3-O- sulphur
The sour albumen of transferase -1 5mL；By it in after 37 DEG C of warm bath 5 minutes, 2ml 3 '-AMP -5 is added '-phosphoric acid；Reaction is 37
Carried out in DEG C, every 10 minutes determination experiment groups and light absorption value of the control group at 400nm, until OD values no longer change；
Sample is boiled 5 minutes, after filtering removing protein, dialysed, concentrated and freezed.
The present invention can effectively obtain the expression of recombinase Heparan sulfate 3-O sulfotransferases, and be used successfully to close
Into Heparan sulfate.
Brief description of the drawings
Fig. 1 expands gel electrophoresis figure for the 3-O- sulfate transferases -1PCR of embodiment one.
Fig. 2 is the gene order of 3-O- sulfate transferases -1 of embodiment one.
Fig. 3 is the contrast before and after the gene order of 3-O- sulfate transferases -1 optimization of embodiment one.
Fig. 4 is the control electrophoretogram after the induced expression of 3-O- sulfate transferases -1 of embodiment one.
Fig. 5 is the control electrophoretogram after the renaturation of 3-O- sulfate transferases -1 of embodiment two.
The 1H-NMR nuclear magnetic resonance map comparison diagrams that Fig. 6 is NSNAH (a) and NSNA3SHp (b) in embodiment two.
Embodiment
The expression of the recombinase Heparan sulfate 3-O sulfotransferases of embodiment one
(1) real-time RT-PCR (RT-PCR)：According to the Rattus norvegicus 3-O- sulfate transferases -1 reported
Gene (GenBank accession number：NO.NM_053391.1 a pair of specific primers) are devised, restriction enzyme is added in primer
III two restriction enzyme sites of enzyme BamH I and Hind.Using Wistar rat brains total serum IgE as template, Real time reverse transcription polymerase is utilized
The amplification of chain reaction (RT-PCR) technology obtains the genetic fragment of 3-O- sulfate transferases -1.By the obtained fragments of PCR and pMD-19-
Carrier T is connected, and is imported in bacillus coli DH 5 alpha competence, is tested with the screening of blue hickie with bacterium colony PCR
Card.As a result such as Fig. 1 (swimming lanes 1：Mark；Swimming lane 2,3：The PCR DNA fragmentation of 3-O- sulfate transferases -1).
There is obvious band from figure it can be found that at 1000bp, and the PCR primer is believed with American National biotechnology
The gene of Rattus norvegicus 3-O- sulfate transferases -1 (936 bp) length of breath center (NCBI) report is basically identical.To further determine that
PCR primer, is transferred to Invitrogen (Shanghai) Trading Co., Ltd. to be sequenced.Sequencing result is shown in Fig. 2.
Utilize software (DNAMAN) contrast sequencing result and the gene order of Rattus norvegicus 3-O- sulfate transferases -1.It can send out
Existing, both homologys are 99%, there is the difference of 5 bases.
(2) codon optimization：Rare codon analysis is carried out to sequence, while entrusting the deep fast limited public affairs of biotechnology in Suzhou
Department carries out codon optimization.Alignment such as Fig. 3 before and after optimization.
(3) express：Gene after codon optimization is connected to pET-28a carriers.Plasmid is imported using hot robin and expressed
In host e. coli BL21 (DE3).After resistance screening, induced using isopropylthiogalactoside (IPTG), make mesh
Protein expression.Ultrasonication is carried out to the cell after induction, liquid is crushed as total protein sample, centrifuging and taking supernatant and precipitation point
Zuo Wei not soluble and insoluble proteins sample, progress polyacrylamide gel electrophoresis (SDS-PAGE).Through polyacrylamide
Gel electrophoresis (SDS-PAGE) identifies that destination protein is mainly present in insoluble proteins sample with inclusion bodies.Such as Fig. 4
Shown (M:Mark；Swimming lane 1:E. coli bl21 (DE3)/pET-28a-3OST-1 total protein of cell without induction；Swimming lane
2:E. coli bl21 (the DE3)/pET-28a-3OST-1 cells induced through 1mmol/L isopropylthiogalactosides (IPTG)
Total protein；Swimming lane 3:E. coli bl21 (the DE3)/pET- induced through 1mmol/L isopropylthiogalactosides (IPTG)
Albumen in 28a-3OST-1 clasmatosis liquid supernatant；Swimming lane 4:Induced through 1mmol/L isopropylthiogalactosides (IPTG)
E. coli bl21 (DE3)/pET-28a-3OST-1 clasmatosis liquid precipitates in albumen).
From fig. 4, it can be seen that compared with the control, the total protein after induction exists obvious between 29-44.3 kilodaltons
Band overstriking.Fusion protein size is analyzed using software (DNAMAN), it is found that the theoretical size of destination protein is 37.3
Kilodalton, and overstriking band is analyzed through software (UN-SCAN-IT gel 6.1) in figure, molecular weight is 37.4 kilodaltons,
Both are of substantially equal.Prove successful expression 3OST-1 albumen.But destination protein is not present in after clasmatosis
In supernatant, but it is present in the precipitation after crushing, illustration purpose albumen is to be present in cell with insoluble inclusion bodies
In.
Application of the embodiment two in synthesis Heparan sulfate
(4) renaturation：For the inclusion body of inactive, it is necessary to it is correctly folded into soluble protein through renaturation.This
Experiment takes dialysis to carry out renaturation.Main method is by solubilization of inclusion bodies in denaturing liquid, and to load bag filter.Dialyzate
The denaturing liquid of the urea containing 6mol/L is initially, 1.5L renaturation solutions are then added with 0.8mL/min speed, gradually reduced with reaching
The purpose of urea content in dialyzate.As a result as shown in Figure 5 (M:Mark；Swimming lane 1:E. coli bl21 without induction
(DE3)/pET-28a-3OST-1 total protein of cell；Swimming lane 2:Induced through 1mmol/L isopropylthiogalactosides (IPTG)
E. coli bl21 (DE3)/pET-28a-3OST-1 total protein of cell；Swimming lane 3:Through 1mmol/L isopropylthiogalactosides
(IPTG) albumen in e. coli bl21 (DE3)/pET-28a-3OST-1 of induction clasmatosis liquid supernatant；Swimming lane 4:Through
E. coli bl21 (DE3)/pET-28a-3OST-1 cells of 1mmol/L isopropylthiogalactosides (IPTG) induction are broken
Albumen in broken liquid precipitate；Swimming lane 5:The e. coli bl21 induced through 1mmol/L isopropylthiogalactosides (IPTG)
(DE3)/pET-28a-3OST-1 clasmatosis liquid precipitates refolded protein).
Fig. 5 results show that destination protein inclusion body is after renaturation, and miscellaneous band is less in sample, and pillar location is correct.
(5) enzyme activity determination：After renaturation terminates, 10000rpm centrifugation 10min take supernatant, as renaturation is successfully soluble
Destination protein.Utilize 3 '-AMP -5 '-phosphosulfate regenerative system progress enzyme activity determination.3-O- sulfate transferases -1
Enzyme activity is defined as the enzyme amount of the interior conversion 1nmol substrates of 1min or 1nmol sulfate groups under conditions of 37 DEG C, pH 7.0.Enzyme activity meter
Calculate formula as follows：
The meaning of each parameter in formula：Abst=5 and Abst=20 reactants when being enzymatic reaction 5min and 20min respectively
The OD of system400Value, ε0Molar extinction coefficient numerical value is 10.5 × 10-3, RT is the reaction time, and [3OST] is the concentration of enzyme, and 1 represents
The volume of reaction system, 1000 represent μm ol to nmol Conversion of measurement unit.
(6) -1 couple of NSNAH of 3-O- sulfate transferases sulphation modification：Reaction cumulative volume is 20ml, in experimental group successively
Add 20mg N- it is deacetylated/heparosan (NSNAH), 8mL 3 '-AMP -5 '-phosphosulfate, the 5mL virtues of sulphation
NSNAH is added without in fragrant sulfotransferase (AST-IV), the albumen 5mL of 3-O- sulfate transferases -1, control group, other are with experiment
Group.By two components in after 37 DEG C of warm bath 5 minutes, 2ml 3 '-AMP -5 is added '-phosphoric acid.Reaction is carried out in 37 DEG C,
Every 10 minutes determination experiment groups and light absorption value of the control group at 400nm, until OD values no longer change.Sample is boiled
5 minutes, filter after removing protein, dialysed, concentrated and freezed, use1H-NMR is detected.As a result it is as shown in Figure 6
(GlcNS3S:The Glucose sulfate amine of 2,3- bis-；GlcNAc:N- acerylglucosamines；GlcA:Gucosamine；GlcNS:3-
Glucose sulfate amine；Ac:Acetyl group).
As a result show, NSNAH occurs in that the obvious Glucose sulfate amine of 2,3- bis- after modification at 3.43ppm
(GlcNS3S) H2 new peaks, show 3- sulphations-N- it is deacetylated/heparosan (NSNA3SHp) of sulphation successfully made
It is standby.And the peak of 2,3- bis- Glucose sulfate amine (GlcNS3S) H1 at 5.50ppm and N- acerylglucosamines (GlcNAc)
H1 closely, significantly can not be told from figure.
Claims (4)
1. the expression of recombinase Heparan sulfate 3-O sulfotransferases, it is characterised in that utilize Wistar rat brain groups
Knit the RNA in source and carry out reverse transcription for template, obtain the complementary DNA sequence of 3-O- sulfate transferases -1, carry out close
Numeral optimizes and cuts off signal peptide；Fragment after optimization is connected with pET-28a plasmids, imports Host Strains e. coli bl21
(DE3) induced expression is carried out in.
2. the method as described in claim 1, it is characterised in that comprise the steps：
(1) real-time RT-PCR (RT-PCR)：According to the gene of Rattus norvegicus 3-O- sulfate transferases -1 reported
(GenBank accession number：NO.NM_053391.1 a pair of specific primers) are devised, restriction enzyme is added in primer
III two restriction enzyme sites of BamH I and Hind；Using Wistar rat brains total RNA as template, it is polymerize using Real time reverse transcription
The amplification of enzyme chain reaction (RT-PCR) technology obtains the genetic fragment of 3-O- sulfate transferases -1；By the obtained fragments of PCR and pMD-19-
Carrier T is connected, and is imported in bacillus coli DH 5 alpha competence, is tested with the screening of blue hickie with bacterium colony PCR
Card；
(2) codon optimization：Rare codon analysis is carried out to sequence, while carrying out codon optimization；
(3) express：Gene after codon optimization is connected to pET-28a carriers, and plasmid is imported into expressive host using hot robin
In e. coli bl21 (DE3)；After resistance screening, induced using isopropylthiogalactoside (IPTG), make purpose egg
White expression；Ultrasonication is carried out to the cell after induction, liquid is crushed as total protein sample, centrifuging and taking supernatant is made respectively with precipitation
For soluble and insoluble proteins sample, polyacrylamide gel electrophoresis (SDS-PAGE) is carried out.
3. application of the recombinase Heparan sulfate 3-O sulfotransferases in synthesis Heparan sulfate, it is characterised in that such as
Obtained described in claim 1 or 2 after expression, carrying out purifying using nickel affinity column removes foreign protein；And using dialysis to forgiving
Body carries out renaturation；By 3 '-AMP -5 '-phosphosulfate regenerative system, Enzyme activity assay is carried out to refolded protein, and should
For substrate N- it is deacetylated/heparosan (NSNAH) of sulphation sulphation modification in.
4. application as claimed in claim 3, it is characterised in that：Method as claimed in claim 2 is obtained after expression, under progress
State step：
(4) renaturation：By solubilization of inclusion bodies in denaturing liquid, and load bag filter；Dialyzate is initially the denaturing liquid of the urea containing 6M,
1.5L renaturation solutions are then added with 0.8mL/min speed；
(5) enzyme activity determination：After renaturation terminates, 10000rpm centrifugation 10min take supernatant, the as successful soluble mesh of renaturation
Albumen；Utilize 3 '-AMP -5 '-phosphosulfate regenerative system progress enzyme activity determination；The enzyme activity of 3-O- sulfate transferases -1
It is defined as the interior enzyme amount for converting 1nmol substrates or 1nmol sulfate groups of 1min under conditions of 37 DEG C, pH 7.0；Enzyme activity calculates public
Formula is as follows：
The meaning of each parameter in formula：Abst=5 and Abst=20 reaction systems when being enzymatic reaction 5min and 20min respectively
OD400Value, ε0Molar extinction coefficient numerical value is 10.5 × 10-3, RT is the reaction time, and [3OST] is the concentration of enzyme, and 1 represents reaction
The volume of system, 1000 represent μm ol to nmol Conversion of measurement unit；
(6) -1 couple of NSNAH of 3-O- sulfate transferases sulphation modification：Reaction cumulative volume is 20ml, is sequentially added in experimental group
The fragrant sulfotransferase (AST-IV) of -5 '-phosphosulfate of 20mg 3 '-AMPs of NSNAH, 8mL, 5mL, the transfer of 3-O- sulfuric acid
The albumen of enzyme -1 5mL；By it in after 37 DEG C of warm bath 5 minutes, 2ml 3 '-AMP -5 is added '-phosphoric acid；Reaction is entered in 37 DEG C
OK, every 10 minutes determination experiment groups and light absorption value of the control group at 400nm, until OD values no longer change；By sample
Boil 5 minutes, after filtering removing protein, dialysed, concentrated and freezed.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1311305A (en) * | 2000-03-02 | 2001-09-05 | 上海博德基因开发有限公司 | New polypeptide-heparan sulfate 3-0-sulfotransferase 19 and polynucleotide for coding such polypeptide |
| US20090035787A1 (en) * | 2007-07-23 | 2009-02-05 | The University Of North Carolina At Chapel Hill | Enzymatic synthesis of sulfated polysaccharides without iduronic acid residues |
| CN102899307A (en) * | 2012-11-19 | 2013-01-30 | 江南大学 | Method for preparing 2-O a-desulfurizing acid enzyme and 3-O a-acidophobe transferase in large scale |
-
2017
- 2017-05-31 CN CN201710396740.5A patent/CN107083372A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1311305A (en) * | 2000-03-02 | 2001-09-05 | 上海博德基因开发有限公司 | New polypeptide-heparan sulfate 3-0-sulfotransferase 19 and polynucleotide for coding such polypeptide |
| US20090035787A1 (en) * | 2007-07-23 | 2009-02-05 | The University Of North Carolina At Chapel Hill | Enzymatic synthesis of sulfated polysaccharides without iduronic acid residues |
| CN102899307A (en) * | 2012-11-19 | 2013-01-30 | 江南大学 | Method for preparing 2-O a-desulfurizing acid enzyme and 3-O a-acidophobe transferase in large scale |
Non-Patent Citations (5)
| Title |
|---|
| GENBANK: "NM_053391.1", 《GENBANK》 * |
| WEIHUA JIN 等: "Increased soluble heterologous expression of a rat brain 3-O-sulfotransferase 1–A key enzyme for heparin biosynthesis", 《PROTEIN EXPRESSION AND PURIFICATION》 * |
| 夏亚穆 等: "肝素和硫酸乙酰肝素前体Heparosan的合成研究进展", 《化学与生物工程》 * |
| 李学亮: "酶法制备heparosan硫酸化衍生物及其活性的研究", 《中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》 * |
| 齐文静: "高活性低分子量肝素的制备及活性研究", 《中国优秀硕士学位论文全文数据库（电子期刊）医药卫生科技辑年期2010/12页码E079-51》 * |
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