CN106754808A - A kind of expression of heparosan sulphation modification enzyme - Google Patents

A kind of expression of heparosan sulphation modification enzyme Download PDF

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CN106754808A
CN106754808A CN201710033024.0A CN201710033024A CN106754808A CN 106754808 A CN106754808 A CN 106754808A CN 201710033024 A CN201710033024 A CN 201710033024A CN 106754808 A CN106754808 A CN 106754808A
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ost
expression
heparosan
phosphate buffer
supernatant
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尹鸿萍
杨轶伟
王莹
黄凤杰
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China Pharmaceutical University
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12N15/09—Recombinant DNA-technology
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    • C12Y—ENZYMES
    • C12Y208/00—Transferases transferring sulfur-containing groups (2.8)
    • C12Y208/02—Sulfotransferases (2.8.2)
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/101—Plasmid DNA for bacteria

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Abstract

The present invention relates to a kind of expression of heparosan sulphation modification enzyme, the method is related to biological technical field, and in particular to new nucleotide sequence, the structure of prokaryotic expression system and preparation method.The artificial synthesized sequences of 6 ost 3 are connected on pET 28a expression vectors, and are transformed into e. coli bl21 competent cell, through IPTG induced expression intracellular soluble proteins.Lysozyme smudge cells, and supernatant is collected, the zymoproteins of 6 OST 3 are obtained through ni-sepharose purification, its specific enzyme activity is 1.53U/mg.The present invention prepares the zymoproteins of 6 OST 3 using Bacillus coli expression, has the advantages that experiment condition is simple, with low cost, and 6 OST 3 are significant for the modification synthesis of macromolecular heparin as toolenzyme.

Description

A kind of expression of heparosan sulphation modification enzyme
Technical field
A kind of expression of heparosan sulphation modification enzyme, the method is related to biological technical field, and in particular to new Nucleotide sequence, the structure of prokaryotic expression system and preparation method.
Background technology
Heparan sulfate (Heparan sulfate, HS) is a kind of poly-sulfated polysaccharide, and it is widely present in cell table Face and extracellular matrix, take part in various bioprocesses, such as embryo development procedure, coagulation process, virus infection Deng.Research finds that Heparan sulfate has multiple biological activities, including anticoagulation, antitumor, antiviral etc., and it is given birth to Thing function is closely related with the sulfate group of modification on polysaccharide.
At present, Heparin Oligosaccharides are mainly prepared by the method for chemical synthesis.But for synthesis pentasaccharides above molecule The heparin of amount, is used alone chemical synthesis very difficult.Enzyme has selectivity and high efficiency, and its reaction condition is gentle, will not Structure to the substrate of macromolecular causes change, therefore can be used for the synthetic modification process of macromolecular heparin.
In the biosynthetic process of Heparan sulfate, what is synthesized first is by glucuronic acid and acetamido glucose The sugar main sugar chain that alternately connection is formed, i.e. heparosan, then carry out modification reaction and obtain the heparin with bioactivity.It is total Six kinds of enzymes take part in the modification reaction of polysaccharide chain：N- sulfuric acid based transferase (N-sulfotransferase), C5 isomerases (C5 Epimerase), 2-O- sulfuric acid based transferase (2-O-sulfotransferase, 2-OST), 3-O- sulfuric acid based transferases (3-O- Sulfotransferase, 3-OST) and 6-O- sulfuric acid based transferase (6-O-sulfotransferase, 6-OST), it is right respectively The different loci of heparosan sugar chain is modified.
The amino acid sequence of the catalysis activity interval albumen by analyzing the 6-OST-3 of mouse of the invention, obtains corresponding Through the new Double-stranded nucleotide sequence (6-ost-3) of codon optimization, using escherichia coli prokaryotic expression system, express and be prepared for The catalysis activity interval albumen of 6-OST-3, it is significant for the modification synthesis of macromolecular heparin as toolenzyme.
The content of the invention
The purpose of the present invention is to use the prokaryotic expression system relatively low to experiment condition requirement, expresses the 6-OST-3 of mouse Catalysis activity interval albumen, obtain solubility expression zymoprotein for heparin modification synthetic reaction.
A kind of expression of heparosan sulphation modification enzyme, comprises the following steps：
(1) 6-ost-3 sequence of the artificial synthesized two ends with NdeI and XhoI restriction enzyme sites, inserts expression after double digestion The site of the NdeI/XhoI of plasmid pET-28a, obtains expression plasmid pET-28a/6-ost-3.
(2) expression plasmid pET-28a/6-ost-3 is transformed into e. coli bl21 competence, obtains recombined engineering Bacterium.
(3) engineering bacteria is seeded in LB culture mediums overnight concussion and cultivate to be connect with 1% as seed liquor, then by seed liquor The amount of kind is seeded in LB fermentation mediums, adds IPTG that 10-12h, centrifugation are induced at 20 DEG C when bacterium solution absorbance is to 0.4-0.6 Collects thalline.
(4) thalline is resuspended in the phosphate buffer of pH 7.4, adds lysozyme smudge cells, and supernatant is collected by centrifugation.
(5) bacteriolyze supernatant obtains 6-OST-3 enzyme egg mortar solution through ni-sepharose purification.
The present invention utilizes escherichia coli prokaryotic expression system, the 6-OST-3 zymoproteins in intracellular solubility expression, and utilizes Ni-sepharose purification obtains enzyme solutions, has the advantages that method is simple, with low cost.Measured through BCA methods, egg mortar contains in zymoprotein solution It is 2.44mg/mL to measure.After measured, its enzyme activity is 1.53U/mg.
Brief description of the drawings
Fig. 1 is the canonical plotting of BCA method determining the protein quantity
Fig. 2 is the enzyme reaction system explanatory diagram in enzyme assay
Fig. 3 is the canonical plotting of product PNP in enzyme assay
Specific embodiment
In conjunction with embodiment, the present invention is elaborated.
Embodiment 1：
(1) structure of recombinant plasmid：6-ost-3 sequence of the artificial synthesized two ends with NdeI and XhoI restriction enzyme sites, warp The site of the NdeI/XhoI of expression plasmid pET-28a is inserted after double digestion, expression plasmid pET-28a/6-ost-3 is obtained.
(2) structure of engineering bacteria：Expression plasmid pET-28a/6-ost-3 is transformed into e. coli bl21 competence, Through kalamycin resistance plate screening, recombination engineering is obtained.
(3) induced expression of engineering bacteria：Engineering bacteria is seeded in LB culture mediums 37 DEG C of concussion and cultivates overnight as seed Liquid, takes 1mL seed liquors and is seeded to 37 DEG C of concussion and cultivates in the LB fermentation mediums of 100mL, is added when bacterium solution absorbance is to 0.6 IPTG induces 10h, 8000rpm centrifugation 10min collects thallines at 20 DEG C.
(4) bacterial cell disruption：Thalline collected by step (3) is resuspended in NaCl, 20mM tricresyl phosphate that 4mL contains 500mM In the phosphate buffer solution of the pH 7.4 of sodium, add the lysozyme of final concentration of 1mg/mL to be stood overnight in 4 DEG C, 12000rpm from Heart 15min collects supernatant.
(5) ni-sepharose purification：By the bacteriolyze supernatant loading of gained in step (4) to nickel post, delayed with the phosphoric acid containing 10mM imidazoles Solution is rushed as cleaning buffer solution, using the phosphate buffer solution containing 100mM imidazoles as elution buffer, eluant component is collected And with phosphate buffer dialysis removal imidazoles, finally obtain enzyme solutions.
Embodiment 2：
(1) structure of recombinant plasmid：6-ost-3 sequence of the artificial synthesized two ends with NdeI and XhoI restriction enzyme sites, warp The site of the NdeI/XhoI of expression plasmid pET-28a is inserted after double digestion, expression plasmid pET-28a/6-ost-3 is obtained.
(2) structure of engineering bacteria：Expression plasmid pET-28a/6-ost-3 is transformed into e. coli bl21 competence, Through kalamycin resistance plate screening, recombination engineering is obtained.
(3) induced expression of engineering bacteria：Engineering bacteria is seeded in LB culture mediums 37 DEG C of concussion and cultivates overnight as seed Liquid, takes 1mL seed liquors and is seeded to 37 DEG C of concussion and cultivates in the LB fermentation mediums of 100mL, is added when bacterium solution absorbance is to 0.4 The IPTG of final concentration of 0.8mmol/L induces 12h, 8000rpm centrifugation 10min collects thallines at 20 DEG C.
(4) bacterial cell disruption：Thalline collected by step (3) is resuspended in NaCl, 20mM tricresyl phosphate that 4mL contains 500mM In the phosphate buffer solution of the pH 7.4 of sodium, add the lysozyme of final concentration of 1mg/mL to be stood overnight in 4 DEG C, 12000rpm from Heart 15min collects supernatant.
(5) ni-sepharose purification：By the bacteriolyze supernatant loading of gained in step (4) to nickel post, delayed with the phosphoric acid containing 10mM imidazoles Solution is rushed as cleaning buffer solution, using the phosphate buffer solution containing 100mM imidazoles as elution buffer, eluant component is collected And with phosphate buffer dialysis removal imidazoles, finally obtain enzyme solutions.
Embodiment 3：
(1) structure of recombinant plasmid：6-ost-3 sequence of the artificial synthesized two ends with NdeI and XhoI restriction enzyme sites, warp The site of the NdeI/XhoI of expression plasmid pET-28a is inserted after double digestion, expression plasmid pET-28a/6-ost-3 is obtained.
(2) structure of engineering bacteria：Expression plasmid pET-28a/6-ost-3 is transformed into e. coli bl21 competence, Through kalamycin resistance plate screening, recombination engineering is obtained.
(3) induced expression of engineering bacteria：Engineering bacteria is seeded in LB culture mediums 37 DEG C of concussion and cultivates overnight as seed Liquid, takes 1mL seed liquors and is seeded to 37 DEG C of concussion and cultivates in the LB fermentation mediums of 100mL, is added when bacterium solution absorbance is to 0.6 The IPTG of final concentration of 1.0mmol/L induces 12h, 8000rpm centrifugation 10min collects thallines at 20 DEG C.
(4) bacterial cell disruption：Thalline collected by step (3) is resuspended in NaCl, 20mM tricresyl phosphate that 4mL contains 500mM In the phosphate buffer solution of the pH 7.4 of sodium, add the lysozyme of final concentration of 1mg/mL to be stood overnight in 4 DEG C, 12000rpm from Heart 15min collects supernatant.
(5) ni-sepharose purification：By the bacteriolyze supernatant loading of gained in step (4) to nickel post, delayed with the phosphoric acid containing 10mM imidazoles Solution is rushed as cleaning buffer solution, using the phosphate buffer solution containing 100mM imidazoles as elution buffer, eluant component is collected And with phosphate buffer dialysis removal imidazoles, finally obtain enzyme solutions.
Embodiment 4：
Determining the protein quantity：
BCA methods are determined, standard curve result such as following table
Sample ID 0 1 2 3 4 5 6 7
Protein concentration (mg/mL) 0 0.025 0.05 0.1 0.2 0.3 0.4 0.5
A562 0.097 0.127 0.154 0.203 0.311 0.416 0.519 0.597
Determined after 10 times of Sample Dilution, its absorbance is 0.352, be computed protein content for 2.44mg/mL.
Embodiment 5：
Enzyme activity determination：
Enzymatic reaction system such as Fig. 2.Blank pipe and testing tube are set, and the MES bufferings that 1mL is separately added into two pipes are molten Liquid (50mmol/L MES, 1mmol/L MgCl2, 1mmol/L MnCl2), add final concentration of 40 μm of ol/L PAP solution, The AST-IV enzyme solutions of the PNPS solution of final concentration of 1mmol/L and final concentration of 1 μ g/ml, in 30 DEG C of concussion reaction 20min. Then the heparosan solution of final concentration of 1mg/mL is added simultaneously, and the 6- of final concentration of 0.1 μ g/ml is added in testing tube OST-3 enzyme solutions, react 30min after 100 DEG C of water-bath 10min terminating reactions, 12000rpm centrifugations under the conditions of 30 DEG C 15min, takes supernatant UV absorption at measure 400nm.6-OST-3 enzyme activity units (U) are defined as：Under these conditions, plus The enzyme amount being catalyzed than blank tube required for generating 1 μm of ol PNP per minute is a unit of activity after entering 6-OST-3.
PNP standard curves result such as following table
pnp(μmol) 0 0.5 1 1.5 2 2.5
A400 0.047 0.058 0.078 0.101 0.123 0.14
After measured, control tube and measure pipe absorbance are respectively 0.157 and 0.174, are computed, the specific enzyme activity of 6-OST-3 It is 1.53U/mg.

Claims (5)

1. a kind of expression of heparosan sulphation modification enzyme, is primarily characterized in that methods described comprises the following steps：
(1) 6-ost-3 sequence of the artificial synthesized two ends with NdeI and XhoI restriction enzyme sites, inserts expression plasmid after double digestion The site of the NdeI/XhoI of pET-28a, obtains expression plasmid pET-28a/6-ost-3.
(2) expression plasmid pET-28a/6-ost-3 is transformed into e. coli bl21 competence, obtains recombination engineering.
(3) engineering bacteria is seeded in LB culture mediums overnight concussion and cultivate as seed liquor, then by seed liquor with appropriate inoculation Amount is seeded in LB fermentation mediums, adds IPTG in induced expression when bacterium solution absorbance to certain limit, and bacterium is collected by centrifugation Body.
(4) thalline is resuspended in the phosphate buffer of pH7.4, adds lysozyme smudge cells, and supernatant is collected by centrifugation.
(5) bacteriolyze supernatant obtains 6-OST-3 zymoprotein solution through ni-sepharose purification.
2. the method according to required by right 1, it is characterized in that being that the 6-ost-3 sequences in step (1) are new artificial synthesized Sequence.
3. the method according to required by right 1, it is characterized in that being that the inoculum concentration in step (3) is 1%.
4. the method according to required by right 1, it is characterized in that being that the inductive condition in step (3) is：Add the end of IPTG dense It is 0.8-1.0mmol/L to spend, and inducing temperature is 20 DEG C, and induction time is 10-12h.
5. the method according to required by right 1, it is characterized in that the cleaning buffer solution for being ni-sepharose purification in step (5) is containing 10mM The phosphate buffer solution of imidazoles, elution buffer is the phosphate buffer solution containing 100mM imidazoles.
CN201710033024.0A 2017-01-13 2017-01-13 A kind of expression of heparosan sulphation modification enzyme Pending CN106754808A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2441984A1 (en) * 2001-03-28 2002-10-10 Massachusetts Institute Of Technology Methods of 6-o-sulfating polysaccharides and 6-o-sulfated polysaccharide preparations
CN104195157A (en) * 2014-08-20 2014-12-10 江苏大学 High-efficiency recombination expression and purification method of biological active peptide in prokaryotic cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2441984A1 (en) * 2001-03-28 2002-10-10 Massachusetts Institute Of Technology Methods of 6-o-sulfating polysaccharides and 6-o-sulfated polysaccharide preparations
CN104195157A (en) * 2014-08-20 2014-12-10 江苏大学 High-efficiency recombination expression and purification method of biological active peptide in prokaryotic cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LIJUAN ZHANG ET AL: "6-O-Sulfotransferase-1 Represents a Critical Enzyme in the Anticoagulant Heparan Sulfate Biosynthetic Pathway", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 *

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Application publication date: 20170531