CN105886491A - Method for displaying human arginase1 on surfaces of escherichia coli - Google Patents
Method for displaying human arginase1 on surfaces of escherichia coli Download PDFInfo
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- CN105886491A CN105886491A CN201610218512.4A CN201610218512A CN105886491A CN 105886491 A CN105886491 A CN 105886491A CN 201610218512 A CN201610218512 A CN 201610218512A CN 105886491 A CN105886491 A CN 105886491A
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Abstract
The invention provides a method for displaying human arginase1 on surfaces of escherichia coli. The method comprises steps as follows: 1) recombinant plasmids for surface display of human arginase1 are constructed; 2) the recombinant plasmids are converted into escherichia coli competent cells, and genetic engineering strains are obtained; 3) the recombinant strains are subjected to shake-flask culture, and the display efficiency and enzyme activity of human arginase1 are detected; 4) L-Arginine (L-arginine, L-Arg) is efficiently converted by means of the recombinant strains for synthesis of L-Ornithine (L-ornithine, L-Orn). Human arginase1 is effectively displayed on surfaces of escherichia coli cells through improved Type V self-transport protein (Antigen 43), and industrial application of human arginase1 is realized more rapidly. By comparison with an original Type V self-transport protein (Antigen 43) display system, the display efficiency and the enzyme activity of human arginase1 are remarkably improved; by comparison with an existing chitin immobilization method, the synthesis cost is reduced, the technological process is shortened, and the purification procedures are simplified.
Description
Technical field
The present invention relates to the sufacing of people source arginase-1 (Human Arginase1), especially by transhipment protein A ntigen 43 surface display method certainly optimized, it is a kind of new thought, new way, the new method that people source Arginase 1 is shown.
Background technology
People source arginase-1 is present among people's liver cell, participates in urea metabolism circulation, and catalyzing hydrolysis L-Arg generates L-Orn and carbamide.Arginase 1 defect, can cause a series of relevant disease, such as cause hypoevolutism, intellectual deficiency, epilepsy and movement disorder.People source arginase-1 or potential clinical application, may be used for treating cancer.
Arginase is difficult to solution expression with high efficiency in escherichia coli, separation and Extraction from liver, is the main source of arginase, but there is the risk carrying various virus.People source arginase-1 achieves secreting, expressing at pichia pastoris phaff, the people source arginase-1 of purification, after chitin embeds, can effectively convert L-Arg and synthesize L-Orn, but, the method, there is the used time longer, preparation process is complicated, in operation, is easily lost the shortcomings such as enzyme work.
Surface of E. coli display technique, has wide practical use, and such as, glutamate decarboxylase is illustrated in surface of E. coli, and recombinant Bacillus coli cells can convert glutamic acid as " immobilized cell factory " and produce pharmaceutical intermediate γ-aminobutyric acid；Nitrilase is illustrated in E. coli cell surface, and recombinant Bacillus coli cells can be as " immobilized cell molecular sieve " resolving chiral compound o-6-mandelic acid；Organophosphor hydrolytic enzyme is shown surface of E. coli, harmful organophosphorus reagent that recombinant Bacillus coli cells exists as " immobilized biological adsorbent " degraded environment；
Escherichia coli are derived from from transhipment protein A ntigen 43 albumen, as anchorin, can be anchored on Bacillus coli cells adventitia by the β-tubbiness of c-terminus, utilize this feature, it is that a class shows the display systems of heterologous protein at surface of E. coli that people have developed.It is made up of three parts from transhipment protein A ntigen 43 albumen, signal peptide, transport protein and c-terminus functional domain.It is reported, the transhipment protein A ntigen 43 certainly of total length, single c-terminus functional domain can be situated between heterologous protein in surface of E. coli displaying, it is possible to realizes surface display and the immobilization thereof of multimeric protein.
Summary of the invention
The purpose of the present invention is to propose to a kind of realize the method that people source arginase-1 is shown at surface of E. coli, it is provided that a kind of brand-new, the method that effective people source arginase-1 is shown.
The present invention is realized in.Design and replace Antigen 43 aminoterminal primary signal peptide with charged polypeptide, and at the aminoterminal design His label of ARG1, it is simple to efficiency is shown in detection；
1) first, design construction recombiant plasmid pET23a/H6ARG1-Ag43(Figure 1WithFigure 2 , PCR primer is shown inTable 2), secondly by the sequence designing the PCR primer electrically charged polypeptide of introducing, (electrically charged polypeptide and aminoacid sequence thereof are shown inTable 1), replace recombiant plasmid pET23a/H by PCR6The primary signal peptide of the Ag43 albumen on ARG1-Ag43, construction recombination plasmid pET23a/H6K6-ARG1-Ag43 and pET23a/H6D6-ARG1-Ag43 (structural representationFigureSeeFigure 1WithFigure 2 , PCR primer is shown inTable 2)。
Table 1Signal peptide and electrically charged polypeptide and aminoacid sequence thereof
Table 2Primer and primer sequence
Primer direction is 5 '-3 '
2) the recombinant plasmid transformed E. coli expression strains Rosetta Blue competent cell that will build, 37 DEG C of quiescent culture are overnight, it is thus achieved that engineering strain；
3) by engineering strain, be inoculated in 100mL LB culture medium, the final concentration of 100 μ g/mL of ampicillin, 37 DEG C in the shaking flask of 200RPM concussion cultivate, OD value is between 0.5～0.6, addition IPTG, final concentration of 1mM, 37 DEG C of inducing culture 8 hours.Then, 12000RPM, 4 DEG C, 10 minutes centrifugal collection thalline, thalline pre-cooling phosphate buffer cleans 3 times, is resuspended in the pre-cooling phosphate buffer of 10mL standby；
4) taking 200 bacterium solution resuspended for μ L respectively, cell, after fluorescent antibody labelling, analyzes primary signal peptide and the efficiency of electrically charged polypeptide display people source arginase-1 with stream Schwann Cells art (Flow Cytometry, FCM)；
5) with Chinard reaction detection L-Orn content, and then the genetic engineering bacterium catalyzed conversion L-Arg synthesis L-Orn enzyme analyzing primary signal peptide and electrically charged polypeptide display people source arginase-1 is lived.Whole reaction system is 1mL, forms as follows: 100 μ L substrate L-Arg, and concentration is 0.2M, 800 μ L bicarbonate buffer (50mM, pH=10) and 100 μ L steps 3) in resuspended standby bacterium solution.Operating procedure is as follows: first, by reaction buffer 40 DEG C of preheatings 5 minutes, then；At 40 DEG C of water-baths, concussion reaction 10 minutes, 12000RPM, 4 DEG C, within 10 minutes, it is centrifuged and collects reacted supernatant, supernatant is terminated reaction in 5 minutes 100 DEG C of heating.Finally, the Chinard response measurement of the L-Orn in supernatant, and it is alive to be converted into corresponding people source arginase-1 enzyme；
6), after measured, select the enzyme the highest engineering strain alive converting L-Arg synthesis L-Orn, then large-scale culture, convert L-Arg and generate L-Orn.Concrete operations are as follows: first, by step 5) in enzyme live the highest engineered strain, renewed vaccination is fresh in 1L LB culture medium, the final concentration of 100 μ g/mL of ampicillin, 37 DEG C of concussion cultivations, OD value is between 0.5～0.6, add IPTG, final concentration of 1mM, 37 DEG C of inducing culture 8 hours.Then, 12000RPM, 4 DEG C, 10 minutes centrifugal collects thalline, thalline pre-cooling PBS 3 times, is resuspended in the pre-cooling bicarbonate buffer of 50mL, adds the final concentration of 200g/L of substrate L-Arg, concussion reaction 14 hours in 40 DEG C of water-baths.Being computed wherein L-Orn content and reach 130g/L, transformation efficiency reaches 95%.
The present invention shows arginase-1, people source, including original Antigen 43 people source arginase-1 display systems, Antigen 43 people source arginase-1 display systems that electrically charged polypeptide optimizes.
The present invention has the advantage that.
The present invention realizes people source arginase-1 cell surface in escherichia coli by people source arginase-1 fusion Antigen 43 albumen and effectively shows.Compared with original Antigen 43 protein display system, hence it is evident that improve people source arginase-1 and show that efficiency and enzyme are lived, compared with existing chitin immobilization method, reduce synthesis cost, shortened process, simplify purification step.
Improving nearly 12 times than the displaying efficiency of original Antigen 43 albumen people source arginase-1, the enzyme of people source arginase-1 is lived and is improved nearly 500 times.
Accompanying drawing explanation
Figure 1、Figure 2For experimental design of the present invention.As schemedShowing, Antigen 43 is from the english abbreviation transporting protein A ntigen 43；ARG1 is the english abbreviation of people source arginase-1；His is the english abbreviation of label protein；D6 is the english abbreviation of six aspartic acids；K6 is the english abbreviation of six lysines.Design is as follows, first, and construction recombination plasmid pET23a/H6ARG1-Ag43, secondly charge residue, six aspartic acids and six lysines replace primary signal peptide, construction recombination plasmid pET23a/H6K6-ARG1-Ag43 and pET23a/H6D6-ARG1-Ag43。
Figure 3The displaying efficiency of people source arginase-1 is shown for stream Schwann Cells art analysis.As schemedShow,Figure AFor negative control result；Figure BFor H6D6The result of the displaying efficiency of people source arginase-1 is shown in-ARG1-Ag43 stream Schwann Cells art analysis；Figure C H6ARG1-Ag43 is the result that the displaying efficiency of people source arginase-1 is shown in stream Schwann Cells art analysis；Figure DFor H6K6-ARG1-Ag43 is the result that the displaying efficiency of people source arginase-1 is shown in stream Schwann Cells art analysis.
Figure 4Show that for Chinard reaction monitoring the enzyme of people source arginase-1 is lived.As schemedShow, H6ARG1-Ag43, H6D6-ARG1-Ag43 and H6K6-ARG1-Ag43 is respectively the enzyme monitoring result alive showing people source arginase-1.
Detailed description of the invention
With example, the present invention is further described below:
Embodiment 1:
The inventive method is utilized to show people source arginase-1 in escherichia coli.First, the recombiant plasmid pET23a/H that will build6ARG1-Ag43 converts competent escherichia coli cell Rosetta Blue bacterial strain, and 37 DEG C of quiescent culture are overnight.Then, picking list bacterium colony, it is inoculated in 100mL LB culture medium, the final concentration of 100 μ g/mL of antibiotics ampicillin, 37 DEG C of concussions cultivate, and OD value is between 0.5～0.6, addition IPTG, final concentration of 1mM, 37 DEG C of inducing culture 8 hours.Then, 12000RPM, 4 DEG C centrifugal collects thalline, thalline pre-cooling PBS 3 times, is finally resuspended in 10mL pre-cooling PBS standby.Then use Chinard response measurement, and it is alive to be converted into corresponding people source arginase-1 enzyme.(Figure 3C,Figure 4)
Embodiment 2
The inventive method is utilized to show people source arginase-1 in escherichia coli.First, the recombiant plasmid pET23a/H that will build6D6-ARG1-Ag43 converts competent escherichia coli cell Rosetta Blue bacterial strain, and 37 DEG C of quiescent culture are overnight.Then, picking list bacterium colony, it is inoculated in 100m L LB culture medium, the final concentration of 100 μ g/mL of antibiotics ampicillin, 37 DEG C of concussions cultivate, and OD value is between 0.5～0.6, addition IPTG, final concentration of 1mM, 37 DEG C of inducing culture 8 hours.Then, 12000RPM, 4 DEG C centrifugal collects thalline, thalline pre-cooling PBS 3 times, is finally resuspended in 10mL pre-cooling PBS standby.Then use Chinard response measurement, and it is alive to be converted into corresponding people source arginase-1 enzyme.(Figure 3B,Figure 4)
Embodiment 3
The inventive method is utilized to show people source arginase-1 in escherichia coli.First, the recombiant plasmid pET23a/H that will build6K6-ARG1-Ag43 converts competent escherichia coli cell Rosetta Blue bacterial strain, and 37 DEG C of quiescent culture are overnight.Then, picking list bacterium colony, it is inoculated in 100mL LB culture medium, the final concentration of 100 μ g/mL of antibiotics ampicillin, 37 DEG C of concussions cultivate, and OD value is between 0.5～0.6, addition IPTG, final concentration of 1mM, 37 DEG C of inducing culture 8 hours.Then, 12000RPM, 4 DEG C centrifugal collects thalline, thalline pre-cooling PBS 3 times, is finally resuspended in 10mL pre-cooling PBS standby.Then use Chinard response measurement, and it is alive to be converted into corresponding people source arginase-1 enzyme.(Figure 3D,Figure 4)
Various sample result analyses
Analyze through stream Schwann Cells art, electrically charged polypeptide 6 × Glu and 6 × Lys can improve the displaying efficiency of Antigen 43 protein display people source arginase-1, the most electrically charged polypeptide 6 × Glu is about 1～2 times of original Antigen 43 protein display people source arginase-1, and wherein 6 × Lys is about 10 times of original Antigen 43 protein display people source arginase-1.Live with Chinard response analysis enzyme, identical result can be obtained, electrically charged polypeptide 6 × Glu and 6 × Lys can improve the enzyme of Antigen43 protein display system demonstration people source arginase-1 and live, the most electrically charged polypeptide 6 × Glu is about 40 times of original Antigen 43 protein display people source arginase-1, and wherein 6 × Lys is about 500 times of original Antigen 43 protein display people source arginase-1., wherein 6 × Lys enzyme is lived and is up to 15U/OD600.The methods of exhibiting that this research optimizes, effectively conversion of substrate L-Arg can synthesize L-Orn, react 14 hours, the substrate L-Arg of 200g/L can be fully converted to L-Orn.
Coli strain Rosetta Blue of the present invention is purchased from Novagen company of the U.S., and different recombiant plasmid is this experimental construction, and analyzing with various reagent is analytical pure.
Claims (1)
1. one kind realizes the method that people source arginase-1 is shown at surface of E. coli, it is characterised in that:
1) first, design construction recombiant plasmid pET23a/H6ARG1-Ag43；Secondly, design PCR primer carries coding-belt
The sequence of charged polypeptide 6 × Lys and 6 × Asp, replaces recombiant plasmid pET23a/H by PCR6Ag43 on ARG1-Ag43
The primary signal peptide of albumen, builds recombinant expression plasmid pET23a/H6K6-ARG1-Ag43 and pET23a/H6D6-
ARG1-Ag43；
2) the recombinant plasmid transformed E. coli expression strains Rosetta Blue competent cell that will build, 37 DEG C of quiescent culture
Overnight, it is thus achieved that engineering strain；
3) by engineering strain, being inoculated in inducing culture in LB culture medium, centrifugal collection bacterial strain, this bacterial strain resists through fluorescence
After body tag, analyze primary signal peptide and electrically charged polypeptide to Ag43 protein display people source arginase-1 by stream Schwann Cells art
Efficiency；
With Chinard reaction detection L-Ornithine content, and then primary signal peptide and electrically charged polypeptide are to Ag43 protein display
The impact that arginase-1 catalyzed conversion L-Arg synthesis L-Orn enzyme in people source is lived；
4), after measured, the enzyme high engineering strain alive converting L-Arg synthesis L-Orn is selected, in large-scale culture,
Thalline is through collecting, and the medium component of remaining is removed in washing, is resuspended in bicarbonate buffer, using L-Arg as substrate, turns
It is combined to L-Orn.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350494A (en) * | 2016-08-29 | 2017-01-25 | 湖北大学 | Methyl parathion hydrolase preparation method with flavin fluorescent protein anchoring and optimization |
| CN106591343A (en) * | 2016-11-29 | 2017-04-26 | 湖北大学 | Method for secretory expression of super-folded green fluorescent protein mediated heterologous protein in escherichia coli |
| CN108676808A (en) * | 2018-05-25 | 2018-10-19 | 厦门大学 | A kind of recombinant plasmid pET28a-Ag43, surface showing plasmid, recombination engineering and its application |
| CN109628436A (en) * | 2019-01-18 | 2019-04-16 | 重庆派金生物科技有限公司 | A kind of preparation method of fixed recombinant human arginase |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106350494A (en) * | 2016-08-29 | 2017-01-25 | 湖北大学 | Methyl parathion hydrolase preparation method with flavin fluorescent protein anchoring and optimization |
| CN106350494B (en) * | 2016-08-29 | 2020-05-19 | 湖北大学 | Method for anchoring and optimally preparing methyl parathion hydrolase by using flavin fluorescent protein |
| CN106591343A (en) * | 2016-11-29 | 2017-04-26 | 湖北大学 | Method for secretory expression of super-folded green fluorescent protein mediated heterologous protein in escherichia coli |
| CN106591343B (en) * | 2016-11-29 | 2020-02-18 | 湖北大学 | Secretory expression method of superfolder green fluorescent protein mediated heterologous protein in escherichia coli |
| CN108676808A (en) * | 2018-05-25 | 2018-10-19 | 厦门大学 | A kind of recombinant plasmid pET28a-Ag43, surface showing plasmid, recombination engineering and its application |
| CN108676808B (en) * | 2018-05-25 | 2021-02-12 | 厦门大学 | Recombinant plasmid pET28a-Ag43, surface display plasmid, recombinant engineering bacterium and application thereof |
| CN109628436A (en) * | 2019-01-18 | 2019-04-16 | 重庆派金生物科技有限公司 | A kind of preparation method of fixed recombinant human arginase |
| CN109628436B (en) * | 2019-01-18 | 2022-03-29 | 重庆派金生物科技有限公司 | Preparation method of fixed recombinant human arginase |
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