CN102617703A - Method for extracting exogenous expressive protein in fermentation bacteria efficiently without cracking - Google Patents
Method for extracting exogenous expressive protein in fermentation bacteria efficiently without cracking Download PDFInfo
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- CN102617703A CN102617703A CN2012100704477A CN201210070447A CN102617703A CN 102617703 A CN102617703 A CN 102617703A CN 2012100704477 A CN2012100704477 A CN 2012100704477A CN 201210070447 A CN201210070447 A CN 201210070447A CN 102617703 A CN102617703 A CN 102617703A
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Abstract
The invention discloses a method for extracting exogenous expressive protein in fermentation bacteria efficiently without cracking. The method comprises the following steps of: centrifuging bacterial liquid of the fermentation bacteria at the temperature of 4 DEG C for 10 to 60 minutes at a centrifugal speed of 6,000 to 10,000 g, and extracting a supernate; suspending centrifugal sediments in a buffer solution to form a mixed solution, stirring the mixed solution warmly at the temperature of between 4 and 25 DEG C for 5 to 60 minutes, and after 24 hours, standing at the temperature of between 4 and 20 DEG C for 1 to 24 hours; centrifuging the bacterial liquid of the fermentation bacteria at the temperature of 4 DEG C for 10 to 60 minutes at the centrifugal speed of 6,000 to 10,000 g; and extracting a supernate, and mixing the two kinds of supernate to be used as a synthetic protein product. According to the method, the expressive protein which is expressed in the technical process of bacterial fermentation and is detained in bacterial cytoplasm space is extracted efficiently on the premise that bacterial cells are not cracked, so that the ingredients of used materials are simple, the operation process is simple and the comprehensive production cost is low.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of method that need not external source expressing protein in the cracking high efficiency extraction fermenting bacterial.
Background technology
The solubility recombinant protein efficiently expresses through bacterium class fermentation process becomes the important means of producing bioprotein in the modern biotechnology.In the biological medicine compound probability, bacterium class fermentation method expressing protein class biological medicine molecule such as humanization therapeutic antibodies fragment, has obtained using widely in actual industrial production in modern times.
Utilization bacterium class fermentation process is produced in the production technique of soluble proteins, after fermentation ends, need the product albumen that the bacterium class produces be separated from zymocyte liquid and purify out, forms product.In separating the proteic process of purified product, the product albumen of significant proportion can be stranded in the tenuigenin space of bacterium.Effectively discharge the external source soluble proteins in the bacterial cytoplasm space, the collection effciency of the soluble proteins that raising bacterium class fermentation method is expressed becomes the significant challenge that improves the related production state of the art and improve fermentation production efficiency.
In the world wide, the researchist has widely bacterium class fermentative prepn recombinant protein technology and attempts, but in the raising of the production efficiency of recombinant protein, runs into many difficulties.In the correlative study, a large amount of trials are arranged with regard to effective problem that discharges the external source soluble proteins in the fermenting bacterial kytoplasm space.For example use the ultrasonic oscillation method; Utilization stain remover cracked method; And the adding additive increases method of bacterial cell membrane permeability or the like in nutrient solution.The common feature of these methods of the prior art is with zymogenic cytoclasis, is stranded in intracytoplasmic product albumen thereby discharge.Ruinate zymophyte also can discharge like nucleic acid in the proteic while of releasing product, carbohydrate, and lipid, other biological macromole such as membranin cause the bacterium fluid viscosity to increase, and influence the purification efficiency and the purity of product albumen.
Summary of the invention
The technical problem that the present invention mainly solves provides a kind of simple to operate; The comprehensive lower method that need not external source expressing protein in the cracking high efficiency extraction fermenting bacterial of production cost; This method can keep the bacterial cell integrity; Bacterial cell is not carried out under the prerequisite of cracking operation, expressed in the high efficiency extraction fermentation using bacteria technological process, be stranded in the foreign protein in the bacterium kytoplasm space.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is: a kind of method that need not external source expressing protein in the cracking high efficiency extraction fermenting bacterial comprises the steps:
1) preparation damping fluid: with sodium ethylene diamine tetracetate 1.0mmol-500mmol, urea 1.0mol-10.0mol, sucrose 0.1mmol-5mmol, Tris alkali 10-100mmol/L, uniform mixing.Add zero(ppm) water to 950ml, stir 30min under the room temperature and clarify to liquid, the pH value of using Tris-Base or Tris-HCl buffer system adjustment solution continues to add zero(ppm) water to 1L, 120 ℃ of autoclaving 20min to 5-8;
(2) with fermenting bacterial bacterium liquid high speed centrifugation 10-60min, centrifugal speed is: 6000-10000g, and centrifuging temperature is 4 ℃, leaves and takes supernatant, is used for downstream purification;
(3) with the centrifugal deposition thing, be suspended in and form mixed solution in the damping fluid, mixed solution at 4-25 ℃ of following mild stirring 5-60min, after 24 hours, under 4-20 ℃, is left standstill 1-24h, subsequent use;
(4) with the fermenting bacterial bacterium liquid high speed centrifugation 10-60min of step (3), centrifugal speed is: 6000g-10000g, 4 ℃; Leave and take supernatant liquid, the supernatant in the combining step (2) carries out downstream purification as the synthetic proteins product.
In preferred embodiment of the present invention, the consisting of of described damping fluid: sodium ethylene diamine tetracetate 1.0mmol-500mmol, urea 1.0mol-10.0mol, sucrose 0.1mmol-5mmol, Tris alkali 10-100mmol and water 0.9-1L.
In preferred embodiment of the present invention, in the described step (3), the blending ratio of settling and damping fluid is 1:5-5:1.
In preferred embodiment of the present invention, described bacterium is the Gram-negative bacteria of exogenous protein expression.
In preferred embodiment of the present invention, described bacterium is intestinal bacteria and subspecies thereof.
The invention has the beneficial effects as follows:
1, the present invention is keeping the bacterial cell integrity; Bacterial cell is not carried out under the prerequisite of cracking operation; Expressed in the high efficiency extraction fermentation using bacteria technological process, be stranded in the foreign protein in the bacterium kytoplasm space, improve the production efficiency of bacterium class fermentative prepn recombinant protein, reduce starting material and time cost in the foreign aid of the unit protein production process simultaneously; Whole process operation is simple, and comprehensive cost is cheap.
2, use protein or the polypeptide product that the present invention extracted can keep the biological activity of height and the integrity of molecular structure, thus help product be further used in pharmaceutical grade protein, laboratory with reagent and reagent raw material and medical diagnosis with reagent and reagent raw material.Extremely application prospects is arranged in the commercial process of recombinant protein microbial expression.
Description of drawings
Fig. 1 is the human protein kinase PKB enzymic activity detection figure that the present invention extracts;
Fig. 2 is that human protein kinase PKB and the cracking process that the present invention extracts extracts human protein kinase PKB enzymic activity detection comparison diagram;
Fig. 3 is the Fab antibody reduced turbidity test comparison figure that the anti-6-His polypeptide of the recombination human source Fab antibody that extracts of the present invention and cracking process extract;
Fig. 4 is the Fab antibody reduced turbidity test comparison figure that the anti-6-His polypeptide of the recombination human source Fab antibody that extracts of the present invention and cracking process extract.
Embodiment
A kind of method that need not external source expressing protein in the cracking high efficiency extraction fermenting bacterial comprises the steps:
(1) preparation damping fluid: with sodium ethylene diamine tetracetate 1.0mmol-500mmol, urea 1.0mol-10.0mol, sucrose 0.1mmol-5mmol, Tris alkali 10-100mmol/L, uniform mixing.Add zero(ppm) water to 950ml, stir 30min under the room temperature and clarify to liquid, the pH value of using Tris-Base or Tris-HCl buffer system adjustment solution continues to add zero(ppm) water to 1L, 120 ℃ of autoclaving 20min to 5-8;
(2) with fermenting bacterial bacterium liquid high speed centrifugation 10-60min, centrifugal speed is: 6000-10000g, and centrifuging temperature is 4 ℃, leaves and takes supernatant, is used for downstream purification;
(3) with the centrifugal deposition thing, be suspended in and form mixed solution in the damping fluid, mixed solution at 4-25 ℃ of following mild stirring 5-60min, after 24 hours, under 4-20 ℃, is left standstill 1-24h, subsequent use;
(4) with the fermenting bacterial bacterium liquid high speed centrifugation 10-60min of step (3), centrifugal speed is: 6000g-10000g, 4 ℃; Leave and take supernatant liquid, the supernatant in the combining step (2) carries out downstream purification as the synthetic proteins product.
In the present invention, consisting of of described damping fluid: sodium ethylene diamine tetracetate 1.0mmol-500mmol, urea 1.0mol-10.0mol, sucrose 0.1mmol-5mmol, Tris alkali 10-100mmol and water 0.9-1L; In the described step (3), the blending ratio of settling and damping fluid is 1:5-5:1; Described bacterium is the Gram-negative bacteria of exogenous protein expression, is preferably intestinal bacteria and subspecies thereof.
The high efficiency extraction of embodiment 1 recombinant human protein's kinases PKB from the fermentation intestinal bacteria.
(1) preparation damping fluid
Preparation contains sodium ethylene diamine tetracetate 450mmol, urea 4mol, and lactic acid 2.0mmol, the distilled water solution 0.95L of Hydrocerol A 2.0mmol and sucrose 2.0mmol at room temperature stirs and clarified to liquid in 30 minutes.After using Tris-Base or Tris-HCl buffer system adjustment pH value to 7.6, adding distil water is to 1.0L, and this is damping fluid for 120 ℃ of autoclaving 20min.
(2) from bacterium liquid, obtain heterogenous expression albumen
With fermenting bacterial bacterium liquid high speed centrifugation 10-60min, centrifugal speed is: 6000-10000g, and centrifuging temperature is 4 ℃, leaves and takes supernatant, is used for downstream purification; With the centrifugal deposition thing, be suspended in the damping fluid that to form bacteria liquid long-pending: the mixed solution of damping fluid volume=1.:1., mixed solution at 4-25 ℃ of following mild stirring 5-60min, after 24 hours, under 4-20 ℃, is left standstill 1-24h, at high speed centrifugation 10-60min, centrifugal speed is: 6000g-10000g, 4 ℃; Leave and take supernatant liquid, merge supernatant, carry out downstream purification as the synthetic proteins product.
(2) the proteic purifying of heterogenous expression
Adopt the cationic exchange prepacked column to carry out purifying.With the speed high speed centrifugation protein extraction product of 10000g 30 minutes, get the PB post dialysed overnight of supernatant to 0.02mM, subsequently to protein solution with the speed of 12000rpm 4 ℃ centrifugal 30 minutes, again through the membrane filtration of 0.22 μ m.Each CMM prepackage ion exchange column uses 0.01mM PB damping fluid balance, adopts the PB balance pillar wash-out foreign protein of 10 times of volume 0.02mM parallel with baseline to the A280 detection line.Use arginic acid salt damping fluid stepwise elution.Detecting eluted protein amount (A280) simultaneously collects each several part albumen and is concentrating the back through the kinase activity detection; Detect and show human protein kinase PKB catalytic activity good (experimental result is seen Fig. 1); Extract the product albumen method with cracking process and compare, the product albumen actual measurement activity that present method is extracted has more obviously raising (experimental result is seen Fig. 2).
High efficiency extraction in the anti-6 His polypeptide Fab antibody fermentation of the embodiment 2 recombination human sources intestinal bacteria.
(1) preparation damping fluid
Preparation contains sodium ethylene diamine tetracetate 500mmol/L; Urea 4.5mol/L; Lactic acid 2.0mmol/L, the distilled water solution 0.95L of Hydrocerol A 2.0mmol/L and sucrose 2.0mmol/L at room temperature stirs and clarified to liquid in 30 minutes; After using Tris-Base or Tris-HCl buffer system pH value to 7.0, adding distil water is to 1L.This is damping fluid for 120 ℃ of autoclaving 20min.
(2) from bacterium liquid, obtain heterogenous expression albumen
With fermenting bacterial bacterium liquid high speed centrifugation 10-60min, centrifugal speed is: 6000-10000g, and 4 ℃, leave and take supernatant, be used for downstream purification; With the centrifugal deposition thing, be suspended in the damping fluid that to form bacteria liquid long-pending: the mixed solution of damping fluid volume=1.:1., mixed solution at 4-25 ℃ of following mild stirring 5-60min, after 24 hours, under 4-20 ℃, is left standstill 1-24h, at high speed centrifugation 10-60min, centrifugal speed is: 6000g-10000g, 4 ℃; Leave and take supernatant liquid, merge supernatant, carry out downstream purification as the synthetic proteins product.
(2) the proteic purifying of heterogenous expression
Adopt the cationic exchange prepacked column to carry out purifying.With the speed high speed centrifugation protein extraction product of 10000g 30 minutes, get the PB post dialysed overnight of supernatant to 0.02mM, subsequently to protein solution with the speed of 12000rpm 4 ℃ centrifugal 30 minutes, again through the membrane filtration of 0.22 μ m.Each CMM prepackage ion exchange column uses 0.01mM PB damping fluid balance, adopts the PB balance pillar wash-out foreign protein of 10 times of volume 0.02mM parallel with baseline to the A280 detection line.Use arginic acid salt damping fluid stepwise elution.Detect eluted protein amount (A280) simultaneously and collect each several part albumen and pass through the kinase activity detection, detect the anti-6 His polypeptide Fab antibody activities in demonstration people source well (experimental result is seen Fig. 3) in concentrated back.Extract the product albumen method with the cracking of conventional cell lysate and compare, the product albumen actual measurement antibody activity that present method is extracted has raising (experimental result is seen Fig. 3).
High efficiency extraction in the anti-GAPDH albumen dAb antibody fragment fermentation of the embodiment 3 reorganization intestinal bacteria.
(1) preparation damping fluid
Preparation contains sodium ethylene diamine tetracetate 500mmol/L; Urea 5mol/L; Lactic acid 2.0mmol/L, the distilled water solution 0.95L of Hydrocerol A 2.0mmol/L and sucrose 2.0mmol/L at room temperature stirs and clarified to liquid in 30 minutes; After using Tris-Base or Tris-HCl buffer system adjustment pH value to 6.5, adding distil water is to 1L.This is damping fluid for 120 ℃ of autoclaving 20min.
(2) from bacterium liquid, obtain heterogenous expression albumen
With fermenting bacterial bacterium liquid high speed centrifugation 10-60min, centrifugal speed is: 6000-10000g, and 4 ℃, leave and take supernatant, be used for downstream purification; With the centrifugal deposition thing, be suspended in the damping fluid that to form bacteria liquid long-pending: the mixed solution of damping fluid volume=1.:1., mixed solution at 4-25 ℃ of following mild stirring 5-60min, after 24 hours, under 4-20 ℃, is left standstill 24h, at high speed centrifugation 10-60min, centrifugal speed is: 6000g-10000g, 4 ℃; Leave and take supernatant liquid, merge supernatant, carry out downstream purification as the synthetic proteins product.
(2) the proteic purifying of heterogenous expression
Adopt the cationic exchange prepacked column to carry out purifying.With the speed high speed centrifugation protein extraction product of 10000g 30 minutes, get the PB post dialysed overnight of supernatant to 0.02mM, subsequently to protein solution with the speed of 12000rpm 4 ℃ centrifugal 30 minutes, again through the membrane filtration of 0.22 μ m.Each CMM prepackage ion exchange column uses 0.01mM PB damping fluid balance, adopts the PB balance pillar wash-out foreign protein of 10 times of volume 0.02mM parallel with baseline to the A280 detection line.Use arginic acid salt damping fluid stepwise elution.Detect eluted protein amount (A280) simultaneously and collect each several part albumen and pass through the kinase activity detection, detect the anti-GAPDH albumen dAb antibody fragment of demonstration active well (experimental result is seen Fig. 4) in concentrated back.Extract the product albumen method with the cracking of conventional cell lysate and compare, the product albumen actual measurement antibody activity that present method is extracted has raising (experimental result is seen Fig. 4).
The above is merely embodiments of the invention; Be not so limit claim of the present invention; Every equivalent structure or equivalent flow process conversion that utilizes specification sheets of the present invention and accompanying drawing content to be done; Or directly or indirectly be used in other relevant technical fields, all in like manner be included in the scope of patent protection of the present invention.
Claims (6)
1. a method that need not external source expressing protein in the cracking high efficiency extraction fermenting bacterial is characterized in that, comprises the steps:
(1) preparation damping fluid: with sodium ethylene diamine tetracetate 1.0mmol-500mmol, urea 1.0mol-10.0mol, sucrose 0.1mmol-5mmol, Tris alkali 10-100mmol/L, uniform mixing.
2. add zero(ppm) water to 950ml, stir 30min under the room temperature and clarify to liquid, the pH value of using Tris-Base or Tris-HCl buffer system adjustment solution continues to add zero(ppm) water to 1L, 120 ℃ of autoclaving 20min to 5-8;
(2) with fermenting bacterial bacterium liquid high speed centrifugation 10-60min, centrifugal speed is: 6000-10000g, and centrifuging temperature is 4 ℃, leaves and takes supernatant, is used for downstream purification;
(3) with the centrifugal deposition thing, be suspended in and form mixed solution in the damping fluid, mixed solution at 4-25 ℃ of following mild stirring 5-60min, after 24 hours, under 4-20 ℃, is left standstill 1-24h, subsequent use;
(4) with the fermenting bacterial bacterium liquid high speed centrifugation 10-60min of step (3), centrifugal speed is: 6000g-10000g, 4 ℃; Leave and take supernatant liquid, the supernatant in the combining step (2) carries out downstream purification as the synthetic proteins product.
3. the method that need not external source expressing protein in the cracking high efficiency extraction fermenting bacterial according to claim 1; It is characterized in that the consisting of of described damping fluid: sodium ethylene diamine tetracetate 1.0mmol-500mmol, urea 1.0mol-10.0mol, sucrose 0.1mmol-5mmol, Tris alkali 10-100mmol and water 0.9-1L.
4. the method that need not external source expressing protein in the cracking high efficiency extraction fermenting bacterial according to claim 1 is characterized in that, in the described step (3), the blending ratio of settling and damping fluid is 1:5-5:1.
5. the method that need not external source expressing protein in the cracking high efficiency extraction fermenting bacterial according to claim 1 is characterized in that described bacterium is the Gram-negative bacteria of exogenous protein expression.
6. the method that need not external source expressing protein in the cracking high efficiency extraction fermenting bacterial according to claim 4 is characterized in that described bacterium is intestinal bacteria and subspecies thereof.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RO93370A2 (en) * | 1985-12-23 | 1987-12-31 | Combinatul Petrochimic,Ro | PROCEDURE FOR OBTAINING BACTERIAL PROTEINS |
| US5424287A (en) * | 1992-02-14 | 1995-06-13 | Laboratoires Om Sa | Extract of bacterial macromolecules, a process for its preparation and a pharmaceutical composition containing the same |
| CN1318374A (en) * | 2000-04-19 | 2001-10-24 | 刘学博 | Preparation of Xinnaomaitong injecta for promoting blood circulation |
| US20030082742A1 (en) * | 1997-07-02 | 2003-05-01 | Astolfi Spartaco F. | Vector for expression of heterologous protein and methods for extracting recombinant protein and for purifying isolated recombinant insulin |
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2012
- 2012-04-14 CN CN2012100704477A patent/CN102617703A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RO93370A2 (en) * | 1985-12-23 | 1987-12-31 | Combinatul Petrochimic,Ro | PROCEDURE FOR OBTAINING BACTERIAL PROTEINS |
| US5424287A (en) * | 1992-02-14 | 1995-06-13 | Laboratoires Om Sa | Extract of bacterial macromolecules, a process for its preparation and a pharmaceutical composition containing the same |
| US20030082742A1 (en) * | 1997-07-02 | 2003-05-01 | Astolfi Spartaco F. | Vector for expression of heterologous protein and methods for extracting recombinant protein and for purifying isolated recombinant insulin |
| CN1318374A (en) * | 2000-04-19 | 2001-10-24 | 刘学博 | Preparation of Xinnaomaitong injecta for promoting blood circulation |
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Application publication date: 20120801 |