CN107102050A - A kind of method for the expression for quantitatively determining the destination protein expressed in microorganism - Google Patents
A kind of method for the expression for quantitatively determining the destination protein expressed in microorganism Download PDFInfo
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- CN107102050A CN107102050A CN201710345562.3A CN201710345562A CN107102050A CN 107102050 A CN107102050 A CN 107102050A CN 201710345562 A CN201710345562 A CN 201710345562A CN 107102050 A CN107102050 A CN 107102050A
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- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
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Abstract
Present method invention belongs to field of protein expression, and in particular to a kind of method for the expression for quantitatively determining the destination protein expressed in microorganism.The method of the expression for quantitatively determining the albumen expressed in microorganism, from the total amount of the quantitatively upper destination protein for effectively determining to express in microorganism, and effectively determine distribution proportion of the destination protein of expression in inclusion body (non-solubility component) and supernatant (soluble component) two parts.Present method invention is simple to operate, and used instrument and equipment is all biology laboratory common instrument.Carry out analyzing proteins expression from quantitative angle, solve existing issue, be following protein expression optimization, albumen amplification produces and isolated and purified offer reliable basis.
Description
Technical field
Present method invention belongs to field of protein expression, is related to a kind of table for the destination protein for quantitatively determining and being expressed in microorganism
Up to the method for situation.
Background technology
In bioprotein research and development and production process, it can all be related to the detection of protein expression situation, including soluble mesh
Albumen expression quantity, and soluble protein and inclusion body ratio etc..And nowadays most of biological scientific research institutions and
Biotech firm, the detection to protein expression situation is mainly qualitatively detected, and qualitative detection effect also complies with one's wishes not to the utmost, than
Such as, protein electrophoresis result is not that very paste is hard to tell, and is exactly very light invisible.Have a strong impact on essential to protein expression situation
Hold, it is impossible to which the expression optimization of accurate instruction following protein, albumen amplification production and follow-up separation and purification of protein work, so as to cause
The waste of time, manpower and resource.
The content of the invention
In view of this, it is an object of the present invention to provide one kind is simple to operate, used instrument and equipment is all biological real
Test room common instrument.Carry out analyzing proteins expression from quantitative angle, solve existing issue, be that following protein expression is excellent
Change, albumen amplification production and the expression feelings for quantitatively determining the destination protein expressed in microorganism that offer reliable basis are provided
The method of condition.
In order to solve the above-mentioned technical problem, the technical scheme is that：
A kind of method for the expression for quantitatively determining the destination protein expressed in microorganism, purpose egg is expressed for one
White microbial inoculum, cumulative volume is VAlwaysMl, it is the absorbance value OD under 600nm illumination in wavelength600For A, from VAlwaysIn take
The bacterium solution volume for testing goal protein expression situation gone out is VInspectionml.So, after the bacterium solution of taking-up removes supernatant through centrifugation,
Bacterial sediment is obtained, the lysis buffer volume added into bacterial sediment is 40VInspectionA ul。
Further, it is 40V by the volumeInspectionThe lysis buffer of A ul mycetome precipitation is mixed, and broken with ultrasound
Broken instrument after bacterial cell disruption, will respectively take 30ul broken bacterium solution in two 1.5ml EP pipes.Add 10ul's in one of EP1 pipes
4* protein sample treatment fluids, the full bacteria liquid sample S for running denaturing protein electrophoresisFull bacterium；Another EP2 pipe is in supercentrifuge
Centrifugation, the supernatant after centrifugation is carefully transferred in another new EP3 pipe, and into EP3 pipes add 10ul 4* albumen samples
Product treatment fluid, the supernatant samples S for running denaturing protein electrophoresisSupernatant；Precipitation remaining in the EP2 pipes of supernatant is removed
As inclusion body, into the EP2 pipes for removed supernatant inclusion body add with supernatant volume identical lysis buffer 30ul,
And 10ul 4* protein sample treatment fluids, the inclusion body sample S for running denaturing protein electrophoresis are added into EP2 pipesInclusion body。
Further, the SFull bacterium, SSupernatant, SInclusion body, the obtained full bacteria liquid sample S without destination protein in the same wayIt is negative
With the pure protein S of known protein concentrationIt is quantitativeTogether with the off denaturing protein electrophoresis of albumen Maker mono-, applied sample amount is all VLoadingul.According to
Protein electrophoresis glue figure result, it can be seen that substantially distribution of the destination protein in supernatant and inclusion body, destination protein is in supernatant
It is many in liquid, or in inclusion body it is more, this is as qualitative.By gel imaging system analyzing proteins running gel figure, it can draw
SFull bacterium, SSupernatant, SInclusion bodyIn respective destination protein amount and S it is quantitative in known protein content ratio so that it is all V to obtain applied sample amountLoading
Ul SFull bacterium, SSupernatant, SInclusion bodyIn the amount of destination protein be respectively mFull bacteriumug,mSupernatantUg, mInclusion bodyUg, wherein mEntirely=mSupernatant+mInclusion body.Therefore,
The ratio of destination protein amount and destination protein total amount in inclusion body is d=m in supernatantSupernatant/mInclusion body, the mesh wherein in supernatant
Albumen be soluble destination protein so that volume be VAlwaysThe total amount M of soluble destination protein in ml microbial inoculumSupernatant
=160mSupernatantVAlwaysA/(3VLoading)ug；Volume is VAlwaysThe concentration of soluble destination protein is D in ml microbial inoculumSupernatant=
160mSupernatantA/(3VLoading)ug/ml.This is quantitative analysis.
Further, the full bacteria liquid sample S without destination proteinIt is negative, i.e., the negative control as experiment, for sentencing
Whether disconnected destination protein has expression in experimental group.
Further, the pure protein S of the known protein concentrationIt is quantitative, i.e., the quantitative scale as experiment, for quantitative mesh
Albumen amount.
Further, the d values are bigger, and the destination protein in supernatant is more, that is, soluble destination protein is more；
Work as d>When 1, the destination protein in supernatant is more than the destination protein in inclusion body, that is, soluble destination protein is more.
Further, the DSupernatant=160mSupernatantA/(3VLoading) ug/ml=60mSupernatantA/(3VLoading) mg/L, represent per L bacterium solutions
In the milligram number containing soluble destination protein.Judge whether the expression of destination protein is considerable with this.
Further, the quantitative analysis is come analyzing proteins running gel figure, for without gel by gel imaging system
The laboratory of imaging system, also can substantially draw the ratio between destination protein and Quantitative Western by naked eyes, so as to draw thick
Corresponding data slightly.
The technology of the present invention effect major embodiment is in the following areas：Present method invention is simple to operate, used instrument
Equipment is all biology laboratory common instrument.Carry out analyzing proteins expression from quantitative angle, existing issue is solved, after being
Continuous protein expression optimization, albumen amplification produces and isolated and purified offer reliable basis.
Brief description of the drawings
Fig. 1 is the protein electrophoresis glue figure of embodiments of the invention；
Fig. 2 is the running gel figure of control group 1 of the invention；
Fig. 3 is the running gel figure of control group 2 of the invention；
Fig. 4 is the running gel figure of control group 3 of the invention.
Embodiment
Below in conjunction with accompanying drawing 1-4, the embodiment to the present invention is described in further detail, so that technical solution of the present invention
It is more readily understood and grasps.
Embodiment
Present method invention is simple to operate, and used instrument and equipment is all biology laboratory common instrument.From qualitative
With carry out analyzing proteins expression in quantitative angle, solve existing issue, be following protein expression optimization, albumen amplification production
And offer reliable basis are provided.
For an induced expression Protein A microbial inoculum, cumulative volume is 50ml, and it is 600nm in wavelength
Illumination under absorbance value OD600 be 4.124, the bacteria liquid for testing goal protein expression situation taken out from V in total
Product is 5ml.So, after the bacterium solution of taking-up removes supernatant through centrifugation, bacterial sediment is obtained, the cracking added into bacterial sediment is delayed
Fliud flushing volume is 824ul.
The volume is mixed for the lysis buffer that 824ul mycetome is precipitated, and broken thalline with Ultrasonic Cell Disruptor
After broken, 30ul broken bacterium solution is respectively taken in two 1.5ml EP pipes.Added in one of EP1 pipes at 10ul 4* protein samples
Manage liquid, the full bacteria liquid sample S for running denaturing protein electrophoresisFull bacterium；Another EP2 pipe is centrifuged in supercentrifuge, after centrifugation
Supernatant be carefully transferred in another new EP3 pipe, and into EP3 pipes add 10ul 4* protein sample treatment fluids, be used for
Run the supernatant samples S of denaturing protein electrophoresisSupernatant；Precipitation as inclusion body remaining in the EP2 pipes of supernatant has been removed, it is past
Inclusion body in the EP2 pipes of supernatant is removed to add and supernatant volume identical lysis buffer 30ul, and has added into EP2 pipes
Enter 10ul 4* protein sample treatment fluids, the inclusion body sample S for running denaturing protein electrophoresisInclusion body。
The SFull bacterium, SSupernatant, SInclusion body, the obtained full bacteria liquid sample S without destination protein in the same wayIt is negativeWith known egg
The pure protein S that white concentration is 1ug/ulIt is quantitativeTogether with the off denaturing protein electrophoresis of albumen Maker mono-, except SIt is quantitativeApplied sample amount be 1ul outside,
Remaining applied sample amount is all 15ul.Protein electrophoresis glue figure result is as shown in Figure 1.
It is by running gel figure qualitative analysis：There are the purposeful protein expression of experimental group, and destination protein ratio bag in supernatant
The destination protein amount contained in body is less.
Quantitative analysis of protein running gel figure is analyzed by Bio-Rad Image Lab gel imaging systems, can be drawn
SFull bacterium：SIt is quantitative=1.27, SSupernatant：SIt is quantitative=0.48, SInclusion body：SIt is quantitative=0.71,
So as to obtain the S that applied sample amount is all 15ulFull bacterium, SSupernatant, SInclusion bodyIn the amount of destination protein be respectively 1.27ug,
0.48ug, 0.71ug, wherein mFull bacteriumIt is substantially equal to mSupernatant+mInclusion body。
Therefore, the ratio of soluble destination protein amount and destination protein total amount in inclusion body is d=mSupernatant/mInclusion body=
0.676, show that soluble protein expression quantity is compared when inclusion body has lacked 32.4%.
So as to total amount of the volume for soluble destination protein in 50ml microbial inoculum
MSupernatant=160mSupernatantVAlwaysA/(3VLoading) ug=352ug；
Volume is VAlwaysThe concentration of soluble destination protein is in ml microbial inoculum
DSupernatant=160mSupernatantA/(3VLoading) ug/ml=7ug/ml=7mg/L.Show under the expression condition, every liter of culture medium
7mg soluble destination protein can be given expression to, such expression does not include especially good, it is necessary to follow-up Optimal Expression condition or excellent
Change plasmid construction.
Experimental example
Experimental group：Completely strictly by disclosed in embodiment, method of the invention is come；
Control group 1：Compared with experimental group, difference is：Not plus quantitative control pure protein；
Control group 2：Compared with experimental group, difference is：Not plus negative control sample and the pure protein of quantitative control；
Control group 3：Compared with experimental group, difference is：Do not come in the present inventive method, but arbitrarily add, do not reorder
Measure albumen.
Control group 1
Sample is handled as stated above, runs electrophoresis, but do not add the pure protein of quantitative control.Running gel figure result is as schemed
Shown in 2.
Contrast negative control group, the purposeful protein expression of experimental group, and purpose in supernatant are can be seen that from running gel figure
Protein content is more more than destination protein amount in inclusion body, shows that soluble destination protein expression quantity is relatively more.But because do not have
There is the pure protein for adding quantitative control, it is impossible to quantitatively determine the absolute magnitude of destination protein, therefore can not accurately determine destination protein
Expression.Because being necessary to add Quantitative Western.
Control group 2
Sample is handled as stated above, runs electrophoresis, but do not add the sample of negative control and the pure protein of quantitative control.
Running gel figure result is as shown in Figure 3.
Because without negative control sample, it is destination protein that can not determine which band.Therefore let alone it is qualitative and
Quantitative analysis.Then the sample plus negative control must be remembered.
Control group 3
In the experimental example, volume is not measured when taking bacterium solution, is not also surveyed after the OD600 of bacterium solution, centrifuged bacterial sediment, with
Meaning has added the lysis buffer of certain volume.Ultrasonic wave is broken after bacterium, and the sample before electrophoresis is not run in processing as stated above, is not calculated
Volume is loaded product and protein sample treatment fluid, simply arbitrarily handles sample, the pure protein of quantitative control is not added, electrophoresis result is as schemed
Shown in 4.
Contrast negative control group, the purposeful protein expression of experimental group are can be seen that from running gel figure.But because processing sample
When do not calculated volume, but arbitrarily sample-adding causes in inclusion body destination protein amount than more than the destination protein in full bacterium, therefore
Expression and distribution situation of the destination protein in supernatant and inclusion body can not be reflected.If even if having added Quantitative Western, Ke Nengtong
The amount that gel analysis system draws destination protein in full bacterium, supernatant and inclusion body is crossed, but because without record-keeping system for electrophoresis sample
Each item data of product, so as to be also the expression that no standard measure calculates soluble destination protein.When therefore preparing electrophoresis Sample
Follow the above method.
The technology of the present invention effect major embodiment is in the following areas：Present method invention is simple to operate, used instrument
Equipment is all biology laboratory common instrument.Carry out analyzing proteins expression from quantitative angle, existing issue is solved, after being
Continuous protein expression optimization, albumen amplification produces and isolated and purified offer reliable basis.
Certainly, it is the representative instance of the present invention above, in addition, the present invention can also have other a variety of specific implementations
The technical scheme of mode, all use equivalent substitutions or equivalent transformation formation, all falls within the scope of protection of present invention.
Claims (8)
1. method that is a kind of qualitative and quantitatively determining destination protein expression in microorganism, it is characterised in that for a table
The microbial inoculum of destination protein is reached, cumulative volume is VAlwaysMl, it is the absorbance value OD under 600nm illumination in wavelength600For
A, from VAlwaysThe bacterium solution volume for testing goal protein expression situation of middle taking-up is VInspectionml.So, the bacterium solution of taking-up is through centrifugation
Go after supernatant, obtain bacterial sediment, the lysis buffer volume added into bacterial sediment is 40VInspectionA ul。
2. method that is as claimed in claim 1 qualitative and quantitatively determining destination protein expression in microorganism, its feature
It is, is 40V by the volumeInspectionThe lysis buffer of A ul mycetome precipitation is mixed, and is broken thalline with Ultrasonic Cell Disruptor
After broken, 30ul broken bacterium solution is respectively taken in two 1.5ml EP pipes.Added in one of EP1 pipes at 10ul 4* protein samples
Manage liquid, the full bacteria liquid sample S for running denaturing protein electrophoresisFull bacterium；Another EP2 pipe is centrifuged in supercentrifuge, after centrifugation
Supernatant be carefully transferred in another new EP3 pipe, and into EP3 pipes add 10ul 4* protein sample treatment fluids, be used for
Run the supernatant samples S of denaturing protein electrophoresisSupernatant；Precipitation as inclusion body remaining in the EP2 pipes of supernatant has been removed, it is past
Inclusion body in the EP2 pipes of supernatant is removed to add and supernatant volume identical lysis buffer 30ul, and has added into EP2 pipes
Enter 10ul 4* protein sample treatment fluids, the inclusion body sample S for running denaturing protein electrophoresisInclusion body。
3. method that is as claimed in claim 2 qualitative and quantitatively determining destination protein expression in microorganism, its feature
It is, the SFull bacterium, SSupernatant, SInclusion body, the obtained full bacteria liquid sample S without destination protein in the same wayIt is negativeIt is dense with known albumen
The pure protein S of degreeIt is quantitativeTogether with the off denaturing protein electrophoresis of albumen Maker mono-, applied sample amount is all VLoadingul.According to protein electrophoresis glue figure
As a result, it can be seen that substantially distribution of the destination protein in supernatant and inclusion body, destination protein is many in supernatant, or
More in inclusion body, this is as qualitative.By gel imaging system analyzing proteins running gel figure, S can be drawnFull bacterium, SSupernatant, SInclusion bodyIn
The ratio of known protein content during the amount and S of respective destination protein are quantitative, so that it is all V to obtain applied sample amountLoadingUl SFull bacterium, SSupernatant,
SInclusion bodyIn the amount of destination protein be respectively mFull bacteriumug,mSupernatantUg, mInclusion bodyUg, wherein mEntirely=mSupernatant+mInclusion body.Therefore, purpose in supernatant
The ratio of destination protein total amount is d=m in protein content and inclusion bodySupernatant/mInclusion body, the destination protein wherein in supernatant is can
Dissolubility destination protein, so that volume is VAlwaysThe total amount M of soluble destination protein in ml microbial inoculumSupernatant=160mSupernatantVAlwaysA/
(3VLoading)ug；Volume is VAlwaysThe concentration of soluble destination protein is D in ml microbial inoculumSupernatant=160mSupernatantA/(3VLoading)ug/
ml.This is quantitative analysis.
4. method that is as claimed in claim 3 qualitative and quantitatively determining destination protein expression in microorganism, its feature
It is, the full bacteria liquid sample S without destination proteinIt is negative, i.e., the negative control as experiment, for judging that destination protein is
It is no to have expression in experimental group.
5. method that is as claimed in claim 3 qualitative and quantitatively determining destination protein expression in microorganism, its feature
It is, the pure protein S of the known protein concentrationIt is quantitative, i.e., the quantitative scale as experiment, the amount for quantitative destination protein.
6. method that is as claimed in claim 3 qualitative and quantitatively determining destination protein expression in microorganism, its feature
It is, the d values are bigger, and the destination protein in supernatant is more, that is, soluble destination protein is more；Work as d>When 1, supernatant
Destination protein in liquid is more than the destination protein in inclusion body, that is, soluble destination protein is more.
7. method that is as claimed in claim 3 qualitative and quantitatively determining destination protein expression in microorganism, its feature
It is, the DSupernatant=160mSupernatantA/(3VLoading) ug/ml=60mSupernatantA/(3VLoading) mg/L, represent per the mesh containing solubility in L bacterium solutions
Albumen milligram number.Judge whether the expression of destination protein is considerable with this.
8. method that is as claimed in claim 3 qualitative and quantitatively determining destination protein expression in microorganism, its feature
It is, the quantitative analysis is come analyzing proteins running gel figure, for the reality without gel imaging system by gel imaging system
Room is tested, the ratio between destination protein and Quantitative Western also can be substantially drawn by naked eyes, so as to draw rough corresponding data.
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Citations (3)
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| CN101386868A (en) * | 2008-09-23 | 2009-03-18 | 复旦大学 | Method for improving expression level of recombinant protein in kluyveromyces |
| CN105044047A (en) * | 2015-06-09 | 2015-11-11 | 天津市南开医院 | Kit for detecting recombinant protein expression and using method thereof |
| CN106591321A (en) * | 2016-11-30 | 2017-04-26 | 浙江农林大学 | Carya cathayensis auxin efflux carrier protein CcPILS gene cloning and expression analysis method |
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2017
- 2017-05-16 CN CN201710345562.3A patent/CN107102050A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101386868A (en) * | 2008-09-23 | 2009-03-18 | 复旦大学 | Method for improving expression level of recombinant protein in kluyveromyces |
| CN105044047A (en) * | 2015-06-09 | 2015-11-11 | 天津市南开医院 | Kit for detecting recombinant protein expression and using method thereof |
| CN106591321A (en) * | 2016-11-30 | 2017-04-26 | 浙江农林大学 | Carya cathayensis auxin efflux carrier protein CcPILS gene cloning and expression analysis method |
Non-Patent Citations (2)
| Title |
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| 杜翠红 等: "乳糖诱导重组人内皮抑素在大肠杆菌中的表达", 《中国医药工业杂志》 * |
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Application publication date: 20170829 |