CN105713888B - A kind of method that the ARG1 immobilization of people source is realized by surface display - Google Patents
A kind of method that the ARG1 immobilization of people source is realized by surface display Download PDFInfo
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- CN105713888B CN105713888B CN201610097606.0A CN201610097606A CN105713888B CN 105713888 B CN105713888 B CN 105713888B CN 201610097606 A CN201610097606 A CN 201610097606A CN 105713888 B CN105713888 B CN 105713888B
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Abstract
The present invention proposes a kind of method that the ARG1 immobilization of people source is realized by surface display.Step is：The aminoterminal designed in ice nucleus formation protein cleavage variant (InaK N) adds signal peptide and electrically charged polypeptide, and in c-terminus fusion people source ARG1, HA labels are designed in c-terminus；Build various recombinant plasmids and convert competent escherichia coli cell respectively, obtain different genes engineered strain；By different strains Shaking culture；Detect people source ARG1 displaying efficiency and enzyme activity；Picking enzyme activity highest bacterial strain mass propgation, Efficient Conversion L arginine, synthesize L ornithines.The present invention is effectively shown by ice nucleus formation protein cleavage variant fusion people source ARG1 in E. coli cell surface, it is achieved thereby that the ARG1 immobilization of people source.Compared with original ice nucleus formation protein display system, hence it is evident that improve people source ARG1 displaying efficiency and enzyme activity, compared with chitin immobilization method, reduce cost, shorten flow, simplify purification step.
Description
Technical field
The present invention relates to people source arginase -1 (Human Arginase1) immobilization technology, especially by
The ice nucleus formation protein display of optimization realizes the method for immobilization, is a kind of new thought, new of people source Arginase 1 immobilization
Approach, new method.
Background technology
People source arginase -1 is present in the form of tripolymer among people's liver cell, is participated in urea metabolism circulation, is urged
Change hydrolysis L-Arg (L-arginine) generation L-Orn (L- ornithines) and urea.Arginase 1 defect, hypoevolutism can be caused,
A series of relevant diseases such as amentia, epilepsy and ataxia, people source arginase -1 can be also used for as drug therapy
Cancer.
Because most of arginase is difficult to realize solution expression with high efficiency in Escherichia coli, extracted from liver
The arginase source main as arginase, but there is may carry various viral risks.People source arginase -1
Effective secreting, expressing can be realized in pichia pastoris phaff, people source arginase -1 of purifying, is embedded by chitin solid
After fixed, effective conversion L-Arg synthesis L-Orn are realized, however, this process for fixation, preparation time is longer, and preparation process is multiple
It is miscellaneous, in operation, the shortcomings of loss part enzyme activity be present.
Surface of E. coli display technique, as a kind of brand-new technique for fixing, have wide practical use, such as, will
Glutamate decarboxylase is illustrated in surface of E. coli, and recombinant Bacillus coli cells can be used as " immobilized cell factory " to convert paddy
Propylhomoserin produces pharmaceutical intermediate γ-aminobutyric acid；Nitrilase is illustrated in E. coli cell surface, recombination bacillus coli is thin
Born of the same parents can be used as the o- 6- mandelic acids of " immobilized cell molecular sieve " resolving chiral compound；Organophosphor hydrolytic enzyme is shown into large intestine bar
Bacterium surface, recombinant Bacillus coli cells are harmful to organophosphorus reagent as existing for " immobilized biological adsorbent " degraded environment；
Sketch prior art ice nucleus formation protein display system.Ice nucleus formation albumen source is in pseudomonad, as grappling
Albumen, Bacillus coli cells outer membrane can be anchored on by phosphatidyl alcohol (GPI), using this feature, people, which have developed, is
A kind of display systems in surface of E. coli displaying heterologous protein.Ice nucleus formation albumen is made up of three parts, aminoterminal function
Domain, height repeat region and c-terminus functional domain.It is reported that the ice nucleus formation albumen of total length, amino acid end shearing mutant, carboxylic
Cardinal extremity shears mutant can mediate heterologous protein to be shown in surface of E. coli with the shearing mutant for removing repetitive sequence.
However, present ice nucleus formation protein display system, it is impossible to the effective surface display and its immobilization for realizing multimeric protein.
By being continuing effort to and improving, people expect to work out efficient, extensive practical ice nucleus formation protein display system, to expire
The various research and production demands of foot.
The content of the invention
The present invention proposes a kind of method that the immobilization of people source arginase -1 is realized by surface display, there is provided a kind of
Brand-new, the method for the immobilization of effective people source arginase -1.
Design ice nucleus formation protein cleavage variant (InaK-N) aminoterminal merge respectively signal peptide (Sec signal peptides and
Tat signal peptides) and electrically charged polypeptide (6 × Lys, 6 × Glu and 6 × Asp), in c-terminus fusion people source arginase -1, and
Its c-terminus designs HA labels, is easy to detection displaying efficiency；
1) first, design PCR primer carries encoded signal peptide Sec, Tat, electrically charged 6 × Lys of polypeptide, 6 × Glu, 6 respectively
× Asp and label protein HA sequence (signal peptide and electrically charged polypeptide and its amino acid sequence are shown in Table 1 and accompanying drawing 1, accompanying drawing 2),
It merges to the aminoterminal in ice nucleus formation protein cleavage variant (InaK-N) respectively by PCR, and people source arginase -1
C-terminus, then two genoid fragments are merged by PCR, constructed containing various containing ice nucleus formation albumen and essence
Propylhomoserin enzyme recombinant expression plasmid；
The Primer and primer sequence are shown in Table 2
The signal peptide of table 1 and electrically charged polypeptide and its amino acid sequence
The Primer of table 2 and primer sequence
A primers direction is 5 ' -3 '
2) the recombinant plasmid transformed E. coli expression strains Rosetta Blue competent cells that will be built, 37 DEG C quiet
Overnight incubation is put, obtains engineering strain；
3) 100mL LB culture mediums are inoculated in by engineering strain, the final concentration of 50 μ g/mL of ampicillin, 37
DEG C concussion and cultivate, between OD values are 0.5~0.6, add IPTG, final concentration of 1mM, 37 DEG C of concussion and cultivates 8 hours.Then,
12000RPM, 4 DEG C, thalline being collected by centrifugation within 10 minutes, thalline is cleaned 2 times with precooling phosphate buffer, removes the culture medium of residual,
It is resuspended in standby in 10mL precooling phosphate buffer；
4) bacterium solution for taking 200 μ L to be resuspended respectively, cell is after fluorescence antibody marks, with stream Schwann Cells art (Flow
Cytometry, FCM) the different signal peptide of analysis and electrically charged polypeptide display people source arginase -1 efficiency；
5) Chinard reaction detection L-Orn contents are used, and then analyze different signal peptides and electrically charged polypeptide display people source
The genetic engineering bacterium catalyzed conversion L-Arg synthesis L-Orn enzyme activity of arginase -1.Whole reaction system is 1mL, and composition is as follows：
It is resuspended in 100 μ L substrate L-Arg, concentration 0.2M, 800 μ L bicarbonate buffers (50mM, pH=10) and 100 μ L steps 3)
Standby bacterium solution.Operating procedure is as follows：First, by reaction buffer 40 DEG C preheat 5 minutes, then；In 40 DEG C of water-baths, concussion
Reaction 10 minutes, 12000RPM, 4 DEG C, reacted supernatant is collected by centrifugation in 10min, and supernatant is terminated for 5 minutes in 100 DEG C of heating
Reaction.Finally, the Chinard response measurements of the L-Orn in supernatant, and it is converted into the corresponding enzyme activity of people source arginase -1；
6) after measured, conversion L-Arg synthesis L-Orn enzyme activity highest engineering strain, extensive training are selected
Support, conversion L-Arg generations L-Orn.Concrete operations are as follows：First, by enzyme activity highest engineered strain, renewed vaccination in step 5)
It is fresh in 1L LB culture mediums, the final concentration of 50 μ g/mL of ampicillin, 37 DEG C of concussion and cultivates, OD values be 0.5~0.6 it
Between, add IPTG, final concentration of 1mM, 37 DEG C of concussion and cultivates 8 hours.Then, 12000RPM, 4 DEG C, bacterium is collected by centrifugation in 10min
Body, thalline precooling PBS 2 times, remove the culture medium of residual, be resuspended in 50mL precooling bicarbonate buffer, add
The final concentration of 200g/L of substrate L-Arg, concussion reaction 16 hours in 40 DEG C of water-baths.One of reaction product L-Orn LC-MS mass spectrums
Identification, confirms as L-Orn reaction products.It is computed wherein L-Orn contents and reaches 130g/L, transformation efficiency reaches 95%.
Present invention displaying people source arginase -1, including original ice nucleus formation albumen people source arginase -1 displaying system
The display systems of ice nucleus formation albumen people source arginase -1 of system, signal peptide and the optimization of electrically charged polypeptide.
The present invention has the advantage that.
The present invention has by ice nucleus formation protein cleavage variant fusion people source arginase -1 in E. coli cell surface
Effect displaying, it is achieved thereby that the immobilization of people source arginase -1.Compared with original ice nucleus formation protein display system, hence it is evident that carry
High people source arginase -1 shows efficiency and enzyme activity, compared with existing chitin immobilization method, reduces synthesis cost, shortens technique
Flow, simplify purification step.
Displaying efficiency than original ice nucleus formation albumen people source arginase -1 improves nearly 3 times, people source arginase -1
Enzyme activity improve it is nearly 3 times.
Brief description of the drawings
Fig. 1, Fig. 2 are experimental design of the present invention.As illustrated, InaK-N shears for ice nucleus formation Argine Monohydrochloride end
The english abbreviation of mutant；ARG1 is the english abbreviation of people source arginase -1；HA is the english abbreviation of label protein；ssMalE
For Sec approach one of which signal peptide english abbreviations；SsTorA is Tat approach one of which signal peptide english abbreviations；D6 is six
The english abbreviation of individual aspartic acid；E6 is the english abbreviation of six glutamic acid；K6 is the english abbreviation of six lysine.Design side
Case is as follows, adds signal peptide in the aminoterminal of ice nucleus formation protein amino-terminus shearing variant respectively, is the signal of Sec approach respectively
The signal peptide of peptide and Tat approach；Charge residue, six aspartic acids, six glutamic acid and six lysines.In c-terminus
People source arginase -1 and label protein HA are added respectively.
Fig. 3 is the displaying efficiency of stream Schwann Cells art analysis displaying people source arginase -1.As illustrated, A is wherein schemed for the moon
Property results of comparison；Figure B is the knot that InaK-N/ARG1 is the displaying efficiency for flowing Schwann Cells art analysis displaying people source arginase -1
Fruit；Figure C is the result that ssMalE-InaK-N/ARG1 is the displaying efficiency for flowing Schwann Cells art analysis displaying people source arginase -1；
Figure D is the result that ssTorA-InaK-N/ARG1 is the displaying efficiency for flowing Schwann Cells art analysis displaying people source arginase -1；Scheme E
For the result that D6-InaK-N/ARG1 is the displaying efficiency for flowing Schwann Cells art analysis displaying people source arginase -1；Figure F is E6-
InaK-N/ARG1 is the result of the displaying efficiency of stream Schwann Cells art analysis displaying people source arginase -1；Figure G is k6-InaK-N/
ARG1 is the result of the displaying efficiency of stream Schwann Cells art analysis displaying people source arginase -1.
Fig. 4 is the enzyme activity that ChinarD reaction monitorings show people source arginase -1.As illustrated, pET23a is unloaded cloudy
Property results of comparison；InaK-N/ARG1, ssMalE-InaK-N/ARG1, ssTorA-InaK-N/ARG1, D6-InaK-N/ARG1,
E6-InaK-N/ARG1 and K6-InaK-N/ARG1 is respectively the enzyme activity monitoring result for showing people source arginase -1.
Fig. 5 is that LC-MS monitoring and displaying people source arginase -1 converts L-Arg synthesis L-Orn.Figure A is L-Arg standard items
LC-MS monitoring results；Figure B is L-Orn standard items LC-MS monitoring results；Figure C is converted product LC-MS monitoring results.
Embodiment
With example, the present invention is further described below：
Embodiment 1：
People source arginase -1 is shown in Escherichia coli using the inventive method.First, InaK- is contained by what is built
N/ARG1 recombinant plasmid transformed competent escherichia coli cell Rosetta Blue bacterial strains, 37 DEG C of quiescent cultures are stayed overnight.Then, choose
Single bacterium colony is taken, is inoculated in 100mL LB culture mediums, the final concentration of 50 μ g/mL of antibiotics ampicillin, 37 DEG C of concussion and cultivates,
Between OD values are 0.5~0.6, IPTG, final concentration of 1mM, 37 DEG C of concussion and cultivates 8 hours are added.Then, 12000RPM, 4 DEG C from
The heart collects thalline, thalline precooling PBS 2 times, removes the culture medium of residual, is finally resuspended in 10mL precooling PBSs
It is standby.Then Chinard response measurements are used, and are converted into the corresponding enzyme activity of people source arginase -1.(Fig. 3, Fig. 4)
Embodiment 2
People source arginase -1 is shown in Escherichia coli using the inventive method.First, contain what is built
SsMalE-InaK-N/ARG1 recombinant plasmid transformed competent escherichia coli cell Rosetta Blue bacterial strains, 37 DEG C of quiescent cultures
Overnight.Then, picking single bacterium colony, is inoculated in 100mL LB culture mediums, the final concentration of 50 μ g/mL of antibiotics ampicillin,
37 DEG C of concussion and cultivates, between OD values are 0.5~0.6, add IPTG, final concentration of 1mM, 37 DEG C of concussion and cultivates 8 hours.Then,
12000RPM, 4 DEG C are collected by centrifugation thalline, thalline precooling PBS 2 times, remove the culture medium of residual, are finally resuspended in 10mL
Precooling PBS is standby.Then Chinard response measurements are used, and are converted into the corresponding enzyme activity of people source arginase -1.
(Fig. 3, Fig. 4)
Embodiment 3
People source arginase -1 is shown in Escherichia coli using the inventive method.First, contain what is built
SsTorA-InaK-N/ARG1 recombinant plasmid transformed competent escherichia coli cell Rosetta Blue bacterial strains, 37 DEG C of quiescent cultures
Overnight.Then, picking single bacterium colony, is inoculated in 100mL LB culture mediums, the final concentration of 50 μ g/mL of antibiotics ampicillin,
37 DEG C of concussion and cultivates, between OD values are 0.5~0.6, add IPTG, final concentration of 1mM, 37 DEG C of concussion and cultivates 8 hours.Then,
12000RPM, 4 DEG C are collected by centrifugation thalline, thalline precooling PBS 2 times, remove the culture medium of residual, are finally resuspended in 10mL
Precooling PBS is standby.Then Chinard response measurements are used, and are converted into the corresponding enzyme activity of people source arginase -1.
(Fig. 3, Fig. 4)
Embodiment 4
People source arginase -1 is shown in Escherichia coli using the inventive method.First, D is contained by what is built6-
InaK-N/ARG1 recombinant plasmid transformed competent escherichia coli cell Rosetta Blue bacterial strains, 37 DEG C of quiescent cultures are stayed overnight.So
Afterwards, picking single bacterium colony, 100mL LB culture mediums, the final concentration of 50 μ g/mL of antibiotics ampicillin, 37 DEG C of concussions are inoculated in
Culture, between OD values are 0.5~0.6, addition IPTG, final concentration of 1mM, 37 DEG C of concussion and cultivates 8 hours.Then, 12000RPM,
4 DEG C are collected by centrifugation thalline, thalline precooling PBS 2 times, remove the culture medium of residual, are finally resuspended in 10mL precoolings PBS and delay
Fliud flushing is standby.Then Chinard response measurements are used, and are converted into the corresponding enzyme activity of people source arginase -1.(Fig. 3, Fig. 4)
Embodiment 5
People source arginase -1 is shown in Escherichia coli using the inventive method.First, E is contained by what is built6-
InaK-N/ARG1 recombinant plasmid transformed competent escherichia coli cell Rosetta Blue bacterial strains, 37 DEG C of quiescent cultures are stayed overnight.So
Afterwards, picking single bacterium colony, 100mL LB culture mediums, the final concentration of 50 μ g/mL of antibiotics ampicillin, 37 DEG C of concussions are inoculated in
Culture, between OD values are 0.5~0.6, addition IPTG, final concentration of 1mM, 37 DEG C of concussion and cultivates 8 hours.Then, 12000RPM,
4 DEG C are collected by centrifugation thalline, thalline precooling PBS 2 times, remove the culture medium of residual, are finally resuspended in 10mL precoolings PBS and delay
Fliud flushing is standby.Then Chinard response measurements are used, and are converted into the corresponding enzyme activity of people source arginase -1.(Fig. 3, Fig. 4)
Embodiment 6
People source arginase -1 is shown in Escherichia coli using the inventive method.First, K is contained by what is built6-
InaK-N/ARG1 recombinant plasmid transformed competent escherichia coli cell Rosetta Blue bacterial strains, 37 DEG C of quiescent cultures are stayed overnight.So
Afterwards, picking single bacterium colony, 100mL LB culture mediums, the final concentration of 50 μ g/mL of antibiotics ampicillin, 37 DEG C of concussions are inoculated in
Culture, between OD values are 0.5~0.6, addition IPTG, final concentration of 1mM, 37 DEG C of concussion and cultivates 8 hours.Then, 12000RPM,
4 DEG C are collected by centrifugation thalline, thalline precooling PBS 2 times, remove the culture medium of residual, are finally resuspended in 10mL precoolings PBS and delay
Fliud flushing is standby.Then Chinard response measurements are used, and are converted into the corresponding enzyme activity of people source arginase -1.(Fig. 3, Fig. 4)
Embodiment 7
Utilize the display systems of ice nucleus formation albumen people source arginase -1 conversion L-Arg synthesis L-Orn of optimization.First,
Contain K by what is built6- InaK-N/ARG1 recombinant plasmid transformed competent escherichia coli cell Rosetta Blue bacterial strains, 37
DEG C quiescent culture is stayed overnight.Then, the fresh strain of renewed vaccination is in 1L LB culture mediums, the final concentration of 50 μ g/ of ampicillin
ML, 37 DEG C of concussion and cultivates, between OD values are 0.5~0.6, add IPTG, final concentration of 1mM, 37 DEG C of concussion and cultivates 8 hours.So
Afterwards, 12000RPM, 4 DEG C, 10min is collected by centrifugation thalline, thalline precooling PBS 2 times, removes the culture medium of residual, is resuspended
In 50mL precooling bicarbonate buffer, the final concentration of 200g/L of substrate L-Arg are added, concussion reaction 16 is small in 40 DEG C of water-baths
When.Reaction product LC-MS Mass Spectrometric Identifications.(Fig. 5)
Various sample result analyses
Analyzed through excessively stream Schwann Cells art, TorA signal peptides, 6 × Glu and 6 × Lys can improve ice nucleus formation protein display
The displaying efficiency of people source arginase -1, it is 1~2 times of left side of original ice nucleus formation protein display people source arginase -1 respectively
The right side, wherein 6 × Lys achieves highest displaying efficiency.MalE signal peptides and 6 × Asp reduce ice nucleus formation protein display people
The displaying efficiency of source arginase -1.With Chinard response analysis enzyme activity, identical result can be obtained, TorA signal peptides, 6
× Glu and 6 × Lys can improve the enzyme activity of ice nucleus formation protein display people source arginase -1, be original ice-nucleus shape respectively
Into 1~2 times or so of the enzyme activity of protein display people source arginase -1, wherein 6 × Lys enzyme activity is up to 11U/OD600。LC-MS
Mass Spectrometric Identification result is shown, the process for fixation originally researched and proposed, effectively can synthesize L-Orn, reaction by conversion of substrate L-Arg
16 hours, 200g/L substrate L-Arg can be fully converted to L-Orn.
Coli strain Rosetta Blue of the present invention are purchased from Novagen companies of the U.S., and different recombinant plasmids is
This experimental construction, analysis are that analysis is pure with various reagents, and LC-MS standard items are chromatographically pure.
Claims (1)
- A kind of 1. method that the immobilization of people source arginase -1 is realized by surface display, it is characterised in that：1) first, design carries the PCR primer of coding-belt charged polypeptide 6 × Lys or 6 × Glu sequence, is melted by PCR The aminoterminal in ice nucleus formation protein cleavage variant InaK-N is closed, design carries the PCR primer of code tag albumen HA sequence, C-terminus in people source arginase -1 is merged by PCR, is then merged two genoid fragments by PCR, Construct the recombinant expression plasmid containing gene K6-InaK-N-ARG1-HA or E6-InaK-N-ARG1-HA；2) the recombinant plasmid transformed E. coli expression strains Rosetta Blue competent cells that will be built, 37 DEG C stand training Support overnight, obtain engineering strain；3) by engineering strain, Fiber differentiation in LB culture mediums is inoculated in, this bacterial strain is thin with streaming after fluorescence antibody marks Born of the same parents' art analyzes influence of the different electrically charged polypeptides to the efficiency of ice nucleus formation protein display people source arginase -1；With Chinard reaction detection L-Ornithine contents, and then different electrically charged polypeptides is analyzed to ice nucleus formation albumen exhibition The catalyzed conversion L-Arg of source arginase -1 that lets others have a look at synthesizes the influence of L-Orn enzyme activity；4) after measured, conversion L-Arg synthesis L-Orn enzyme activity highest engineering strain, large-scale culture, bacterium are selected For body through collecting, the medium component of remaining is removed in washing, is resuspended in bicarbonate buffer, and using L-Arg as substrate, conversion is closed Into L-Orn.
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Inventor after: Ma Lixin Inventor after: Zhang Zhen Inventor after: Wang Yaping Inventor after: Tang Rongxing Inventor before: Zhang Zhen Inventor before: Yi Li Inventor before: Ma Lixin |
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| GR01 | Patent grant | ||
| GR01 | Patent grant |