CN109988803A - A kind of fermentation process of efficient production recombination human serum albumin - Google Patents
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Abstract
The present invention relates to a kind of fermentation process of efficiently production recombination human serum albumin, using Pichia Pastoris as production bacterial strain, pass through the optimization of the various aspects conditions such as temperature, pH, dissolved oxygen level, methanol concentration, medium component to its growth period and induction period, the expression quantity of host cell destination protein recombination human serum albumin (rHSA) is greatly improved, it is avoided in fermentation liquid simultaneously containing excessive yeast cells fragment, is conducive to downstream purification process.By optimization, fermentation medium and the more existing medium component of Fiber differentiation based component are simple, and economic cost can be effectively reduced.The present invention effectively shortens induction period, and entire fermentation process only needs to obtain the recombination human serum albumin that expression quantity is 25.3g/L by 92-116h, has higher production efficiency compared with the prior art, can satisfy huge market demand.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of fermentation side of efficiently production recombination human serum albumin
Method.
Background technique
Human serum albumins (HSA) has played various effects in the clinical treatment of recent decades.Currently, HSA
Production technology using human plasma as raw material, with chilled alcohol precipitation method be the main means of production.But human plasma itself
The risk that supply gap, extremely complex blood plasma source and blood disease are propagated through medicine, all seriously limits the industrialization of HSA
Production.
By the several years, scientific research personnel successfully realizes recombination human serum albumin (rHSA) using a variety of expression systems
Expression.For other expression systems, expression quantity of the HSA in Pichia pastoris is higher, lower production costs, the separation in later period
Purifying process is also more mature, therefore becomes and be most hopeful to realize medicinal HSA industrialization and commodity.Studies have shown that foreign protein exists
Expression quantity in pichia yeast expression system is influenced by series of factors, such as the foreign gene self character and strain of upstream
Characteristic and the culture environment factor in downstream, the addition of auxiliary expression object and the zymotechnique including feed profile etc..
But there is a common problem in correlative study, i.e. the induction time of methanol is too long (generally more than 200 hours), directly
Lead to the low of rHSA production efficiency.Such as the method for patent CN200510068171 fermenting and producing recombinant human serum albumin HSA
360 hours of middle needs could complete entire fermentation process, obtain recombination human serum albumin.We need to improve hair at this stage
Ferment production efficiency, to meet huge market demand.
Summary of the invention
In order to solve the above problems existing in the present technology, the present invention provides a kind of efficiently production recombinant human serum albumin eggs
White fermentation process, using Pichia Pastoris as production bacterial strain, by temperature to its growth period and induction period,
Host cell purpose egg is greatly improved in the optimization of the various aspects condition such as pH, dissolved oxygen level, methanol concentration, medium component
The expression quantity of white HSA.
The technical scheme adopted by the invention is as follows:
(1) rHSA GS115 engineering bacterium solution is taken, switching is shaken on the first YPD culture medium of 900-1000 times of bacterium solution volume
16-18h is cultivated, level-one bacterium solution is obtained, the level-one bacterium solution is continued to transfer and is trained in the 2nd YPD of 900-1000 times of bacterium solution volume
Shake culture 16-18h on base is supported, it is spare to obtain seed liquor；
(2) it being incorporated as fermenting in the fermentation medium that its volume is 4-6 times into seed liquor, temperature is 27-29 DEG C,
PH is 5.5-6.0, and revolving speed is set as 200-800rpm, and dissolved oxygen level is maintained at 19%-21%, obtains fermentation liquid；
(5) when dissolved oxygen level is substantially increased, into fermentation liquid, continuous flow adds concentration to be the glycerol feed supplement liquid of 8-12g/L,
Obtain feed supplement post-fermentation liquid；
(6) when dissolved oxygen level is substantially increased again, continuous flow adds methanol induction liquid and helper-inducer agent to be induced, first
The concentration of alcohol is maintained at 8-12ml/L, and adjusting pH is 5.9-6.1, and temperature is 20-23 DEG C, sends out after being induced after induction 60-80h
Zymotic fluid；
(5) by freezen protective after induction post-fermentation liquid centrifugation, packing to get the hair containing recombination human serum albumin
Zymotic fluid.
Further, in step (1), the preparation method of the first the YPD culture medium and the 2nd YPD culture medium are as follows: take pancreas
Peptone 20g, yeast extract 10g are dissolved in deionized water and are settled to 900mL, 121 DEG C high pressure steam sterilization 20 minutes, to
In the glucose 100mL that 200g/L is added in aseptic superclean bench after cooling.
Further, in step (1), the cultivation temperature is 28-31 DEG C.
Further, in step (2), the fermentation medium contains the yeast extract and 8-12g/L of 23-35g/L
Glycerol.
Further, in step (3), the PTM containing 10-12ml/L in the glycerol feed supplement liquid1With the glycerol of 10g/L.
Further, in step (4), the stream using gas-chromatography off-line monitoring method control methanol adds.
Further, in step (4), the PTM containing 10-12ml/L in the methanol induction liquid1With 1% volume fraction
Methanol.
Further, in step (3) and step (4), the PTM1Preparation method are as follows: take Salzburg vitriol 6g, iodine
Change sodium 0.08g, monohydrate acid magnesium 3g, two molybdic acid hydrate sodium 0.2g, boric acid 0.02g, cobalt chloride 0.5g, zinc chloride 20g, seven water
Ferric sulfate 65g, biotin 0.2g and sulfuric acid 5mL are closed, deionized water is dissolved in together and is settled to 1L.
Further, in step (4), the helper-inducer agent include the vitamin C of 10mM, volume fraction be 0.5%
Casein hydrolysate, 1g/L alanine, 1g/L lysine, 1g/L leucine, 1g/L glutamic acid.
Further, in step (5), the centrifugation is centrifuged 5-15min at 2-4 DEG C with the revolving speed of 7000-8000rpm,
The temperature of the freezen protective is subzero 80 DEG C to subzero 20 DEG C.
Further, the rHSA GS115 engineering bacteria is passed through the bacterial strain screening and gene optimization of many years, benefit by applicant
It is host cell with pichia pastoris yeast GS115 (Pichia pastoris GS115, ATCC 20864) bacterial strain, will optimizes
The human serum albumin gene (from people's liver cell) for being suitable for yeast strain high efficient expression afterwards is by vector construction, matter
Grain linearisation and etc., it is inserted into competence GS115 cell, is screened by MD Screening of Media and G418, finally obtain one plant
The gene engineering microzyme of height expression rHSA, shaking flask level reach 2.5-4mg/ml.
The invention has the benefit that
1, by optimization, fermentation medium and the more existing medium component of Fiber differentiation based component are simple, can effectively drop
Low economic cost, and can also be generated on this basis compared with the higher recombination human serum albumin expression quantity of the prior art.
2, optimize the key factors such as fermentation process and temperature, dissolved oxygen level, feed supplement amount in Induction Process, so that recombined human
Sero-abluminous yield greatly promotes.
3, the prior art cannot be effectively prevented methanol feeding excess by the stream added-time that dissolved oxygen controls methanol, herein by
The stream of gas-chromatography offline inspection control methanol, which adds, effectively prevents methanol feeding excess.
4, the present invention effectively shortens induction period, and fermentation process of the present invention is only needed by 92-116h, can
The recombination human serum albumin that expression quantity is 25.3g/L is obtained, and when the prior art generally needs induction in 200 hours or more
Between, and expression quantity highest also only reaches 15g/L, therefore the present invention has higher production efficiency compared with the prior art.
5, the present invention is avoided in fermentation liquid and was contained by the condition optimizing in culture medium and fermentation and Induction Process
More yeast cells fragments is conducive to downstream purification process.
Specific embodiment
To make the object, technical solutions and advantages of the present invention clearer, technical solution of the present invention will be carried out below
Detailed description.Obviously, described embodiment is only a part of the embodiments of the present invention, instead of all the embodiments.
Based on the embodiments of the present invention, those of ordinary skill in the art obtained institute without making creative work
There is other embodiment, belongs to the range that the present invention is protected.
Embodiment 1
A kind of fermentation process of efficiently production recombination human serum albumin is present embodiments provided, specific processing method includes
Following steps:
(1) rHSA GS115 engineering bacterium solution is taken, is transferred in shake culture on the first YPD culture medium of 900 times of bacterium solution volumes
16h, cultivation temperature are 28 DEG C, obtain level-one bacterium solution, the level-one bacterium solution is continued to transfer in the second of 900 times of bacterium solution volumes
Shake culture 16h, it is spare to obtain seed liquor on YPD culture medium；
The preparation method of the first and second YPD culture medium are as follows: tryptone 20g, yeast extract 10g is taken to be dissolved in
Deionized water is simultaneously settled to 900mL, 121 DEG C high pressure steam sterilization 20 minutes, after cooling in being added in aseptic superclean bench
The glucose 100mL of 200g/L.
(2) it is incorporated as fermenting in the fermentation medium that its volume is 4 times into seed liquor, temperature is 27 DEG C, and pH is
5.5, revolving speed is set as 200rpm, and dissolved oxygen level is maintained at 19%, obtains fermentation liquid；The fermentation medium contains 23g/L's
The glycerol of yeast extract and 8g/L.
(3) when dissolved oxygen level is substantially increased, into fermentation liquid, continuous flow adds concentration to be the glycerol feed supplement liquid of 8g/L, obtains
Feed supplement post-fermentation liquid；Contain the PTM of 10ml/L in glycerol feed supplement liquid1With the glycerol of 10g/L；Take Salzburg vitriol 6g, iodate
Sodium 0.08g, monohydrate acid magnesium 3g, two molybdic acid hydrate sodium 0.2g, boric acid 0.02g, cobalt chloride 0.5g, zinc chloride 20g, seven hydrations
Ferric sulfate 65g, biotin 0.2g and sulfuric acid 5mL are dissolved in deionized water together and are settled to 1L, obtain PTM1。
(4) when dissolved oxygen level is substantially increased again, continuous flow adds methanol induction liquid and helper-inducer agent to be induced, first
The concentration of alcohol is maintained at 8ml/L, the PTM containing 12ml/L in methanol induction liquid1With the methanol of 1% volume fraction, using gas phase
The stream of chromatography off-line monitoring method control methanol induction liquid adds, and adjusting pH is 5.9, and temperature is 20 DEG C, after obtaining induction after induction 60h
Fermentation liquid；Helper-inducer agent include the vitamin C of 10mM, volume fraction be 0.5% casein hydrolysate, 1g/L alanine,
1g/L lysine, 1g/L leucine, 1g/L glutamic acid.
(5) the induction post-fermentation liquid is centrifuged 5 minutes at 2 DEG C with the revolving speed of 7000rpm, after packing freezen protective in
To get the fermentation liquid containing recombination human serum albumin in subzero 20 DEG C.
Embodiment 2
A kind of fermentation process of efficiently production recombination human serum albumin is present embodiments provided, specific processing method includes
Following steps:
(2) rHSA GS115 engineering bacterium solution is taken, is transferred in shake culture on the first YPD culture medium of 1000 times of bacterium solution volumes
18h, cultivation temperature are 31 DEG C, obtain level-one bacterium solution, the level-one bacterium solution is continued to transfer in the second of 1000 times of bacterium solution volumes
Shake culture 18h, it is spare to obtain seed liquor on YPD culture medium；
The preparation method of the first and second YPD culture medium are as follows: tryptone 20g, yeast extract 10g is taken to be dissolved in
Deionized water is simultaneously settled to 900mL, 121 DEG C high pressure steam sterilization 20 minutes, after cooling in being added in aseptic superclean bench
The glucose 100mL of 200g/L.
(2) seed liquor being added in the fermentation medium that volume is 6 times and is fermented, temperature is 29 DEG C, pH 6.0,
Revolving speed is set as 200-800rpm, and dissolved oxygen level is maintained at 21%, obtains fermentation liquid；The fermentation medium contains 35g/L's
The glycerol of yeast extract and 12g/L.
(3) when dissolved oxygen level is substantially increased, into fermentation liquid, continuous flow adds concentration to be the glycerol feed supplement liquid of 12g/L, obtains
To feed supplement post-fermentation liquid；Contain the PTM of 12ml/L in glycerol feed supplement liquid1With the glycerol of 10g/L；Take Salzburg vitriol 6g, iodine
Change sodium 0.08g, monohydrate acid magnesium 3g, two molybdic acid hydrate sodium 0.2g, boric acid 0.02g, cobalt chloride 0.5g, zinc chloride 20g, seven water
Ferric sulfate 65g, biotin 0.2g and sulfuric acid 5mL are closed, deionized water is dissolved in together and is settled to 1L, obtain PTM1。
(4) when dissolved oxygen level is substantially increased again, continuous flow adds methanol induction liquid and helper-inducer agent to be induced, first
The concentration of alcohol is maintained at 12ml/L, the PTM containing 12ml/L in methanol induction liquid1With the methanol of 1% volume fraction, using gas phase
The stream of chromatography off-line monitoring method control methanol induction liquid adds, and adjusting pH is 6.1, and temperature is 23 DEG C, after obtaining induction after induction 80h
Fermentation liquid；Helper-inducer agent include the vitamin C of 10mM, volume fraction be 0.5% casein hydrolysate, 1g/L alanine,
1g/L lysine, 1g/L leucine, 1g/L glutamic acid.
(5) the induction post-fermentation liquid is centrifuged 15 minutes at 4 DEG C with the revolving speed of 8000rpm, freezen protective after packing
To get the fermentation liquid containing recombination human serum albumin in subzero 80 DEG C.
Embodiment 3
A kind of fermentation process of efficiently production recombination human serum albumin is present embodiments provided, specific processing method includes
Following steps:
(1) rHSA GS115 engineering bacterium solution is taken, is transferred in shake culture on the first YPD culture medium of 950 times of bacterium solution volumes
17h, cultivation temperature are 30 DEG C, obtain level-one bacterium solution, the level-one bacterium solution is continued to transfer in the second of 950 times of bacterium solution volumes
Shake culture 17h, it is spare to obtain seed liquor on YPD culture medium；
The preparation method of the first and second YPD culture medium are as follows: tryptone 20g, yeast extract 10g is taken to be dissolved in
Deionized water is simultaneously settled to 900mL, 121 DEG C high pressure steam sterilization 20 minutes, after cooling in being added in aseptic superclean bench
The glucose 100mL of 200g/L.
(2) it is incorporated as fermenting in the fermentation medium that its volume is 5 times into seed liquor, temperature is 28 DEG C, and pH is
5.7, revolving speed is set as 500rpm, and dissolved oxygen level is maintained at 20%, obtains fermentation liquid；The fermentation medium contains 29g/L's
The glycerol of yeast extract and 10g/L.
(3) when dissolved oxygen level is substantially increased, into fermentation liquid, continuous flow adds concentration to be the glycerol feed supplement liquid of 10g/L, obtains
To feed supplement post-fermentation liquid；Contain the PTM of 11ml/L in glycerol feed supplement liquid1With the glycerol of 10g/L；Take Salzburg vitriol 6g, iodine
Change sodium 0.08g, monohydrate acid magnesium 3g, two molybdic acid hydrate sodium 0.2g, boric acid 0.02g, cobalt chloride 0.5g, zinc chloride 20g, seven water
Ferric sulfate 65g, biotin 0.2g and sulfuric acid 5mL are closed, deionized water is dissolved in together and is settled to 1L, obtain PTM1。
(4) when dissolved oxygen level is substantially increased again, continuous flow adds methanol induction liquid and helper-inducer agent to be induced, first
The concentration of alcohol is maintained at 10ml/L, the PTM containing 11ml/L in methanol induction liquid1With the methanol of 1% volume fraction, using gas phase
The stream of chromatography off-line monitoring method control methanol induction liquid adds, and adjusting pH is 6.0, and temperature is 22 DEG C, after obtaining induction after induction 70h
Fermentation liquid；Helper-inducer agent include the vitamin C of 10mM, volume fraction be 0.5% casein hydrolysate, 1g/L alanine,
1g/L lysine, 1g/L leucine, 1g/L glutamic acid.
(5) the induction post-fermentation liquid is centrifuged 10 minutes at 3 DEG C with the revolving speed of 7500rpm, freezen protective after packing
To get the fermentation liquid containing recombination human serum albumin in subzero 50 DEG C.
Comparative example 1
Comparative example 1 is only that the fermented and cultured that the ingredient of fermentation medium is different, in this comparative example from the difference of embodiment 3
The composition of base includes: the yeast basic nitrogen of the tryptone of 40g/L, the yeast extract of 29g/L, the glycerol of 10g/L, 3.4g/L
Base, the ammonium sulfate of 10g/L, the biotin powder of 0.4mg/L, the potassium dihydrogen phosphate of 12g/L, 2.3g/L dipotassium hydrogen phosphate.
Comparative example 2
Comparative example 1 is only that the ingredient of inducible system is different from the difference of embodiment 3, does not add auxiliary in this comparative example and lures
Lead agent.
Comparative example 3
The difference of comparative example 1 and embodiment 3 is only that the temperature of the fermentation in step (2) is 22 DEG C, pH 5.0, dissolved oxygen
Level is 10%.
Comparative example 4
The difference of comparative example 1 and embodiment 3 is only that the temperature of the induction in step (4) is 28 DEG C, pH 7.0, dissolved oxygen
Level is 30%.
Experimental example
The comparison of fermentation process described in embodiment 3 and fermentation process described in comparative example 1-4
Dry cell weight, recombinant human serum albumin egg in fermentation liquid obtained by fermentation process described in Example 3 and comparative example 1-4
White yield is measured, and is compared, and the results are shown in Table 1.
| Group | Dry cell weight (g DCW/L) | Recombination human serum albumin expression quantity (g/L) |
| Embodiment 3 | 193 | 25.3 |
| Comparative example 1 | 189 | 10.7 |
| Comparative example 2 | 191 | 8.3 |
| Comparative example 3 | 123 | 7.5 |
| Comparative example 4 | 187 | 5.1 |
The cell weight and destination protein of described in embodiment 3 it can be seen from the data in upper table fermentation process production
Expression quantity is above comparative example 1-4, also illustrates by ingredient, fermentation and the induction rank to fermentation medium and induced medium
The optimization of the temperature, pH, dissolved oxygen level etc. of section, fermentation process of the present invention can greatly promote destination protein expression
Amount.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain
Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be based on the protection scope of the described claims.
Claims (10)
1. a kind of fermentation process of efficiently production recombination human serum albumin, which comprises the steps of:
(1) rHSA GS115 engineering bacterium solution is taken, is transferred in shake culture on the first YPD culture medium of 900-1000 times of bacterium solution volume
16-18h obtains level-one bacterium solution, and the level-one bacterium solution is continued to transfer in the 2nd YPD culture medium of 900-1000 times of bacterium solution volume
Upper shake culture 16-18h, it is spare to obtain seed liquor；
(2) it is incorporated as fermenting in the fermentation medium of 4-6 times of its volume into seed liquor, temperature is 27-29 DEG C, and pH is
5.5-6.0, revolving speed are set as 200-800rpm, and dissolved oxygen level is maintained at 19%-21%, obtain fermentation liquid；
(3) when dissolved oxygen level is substantially increased, into fermentation liquid, continuous flow adds concentration to be the glycerol feed supplement liquid of 8-12g/L, obtains
Feed supplement post-fermentation liquid；
(4) when dissolved oxygen level is substantially increased again, continuous flow adds methanol induction liquid and helper-inducer agent to be induced, methanol
Concentration is maintained at 8-12ml/L, and adjusting pH is 5.9-6.1, and temperature is 20-23 DEG C, obtains induction post-fermentation after inducing 60-80h
Liquid；
(5) by freezen protective after induction post-fermentation liquid centrifugation, packing to get the fermentation containing recombination human serum albumin
Liquid.
2. a kind of fermentation process of efficiently production recombination human serum albumin according to claim 1, which is characterized in that step
Suddenly in (1), the preparation method of the first the YPD culture medium and the 2nd YPD culture medium are as follows: take tryptone 20g, yeast extract
10g is dissolved in deionized water and is settled to 900mL, 121 DEG C high pressure steam sterilization 20 minutes, after cooling be added 200g/L Portugal
Grape sugar 100mL.
3. a kind of fermentation process of efficiently production recombination human serum albumin according to claim 1, which is characterized in that step
Suddenly in (1), the cultivation temperature is 28-31 DEG C.
4. a kind of fermentation process of efficiently production recombination human serum albumin according to claim 1, which is characterized in that step
Suddenly in (2), the fermentation medium contains the yeast extract of 23-35g/L and the glycerol of 8-12g/L.
5. a kind of fermentation process of efficiently production recombination human serum albumin according to claim 1, which is characterized in that step
Suddenly in (3), the PTM containing 10-12ml/L in the glycerol feed supplement liquid1With the glycerol of 10g/L.
6. a kind of fermentation process of efficiently production recombination human serum albumin according to claim 1, which is characterized in that step
Suddenly in (4), the stream using gas-chromatography off-line monitoring method control methanol adds.
7. a kind of fermentation process of efficiently production recombination human serum albumin according to claim 1, which is characterized in that step
Suddenly in (4), the PTM containing 10-12ml/L in the methanol induction liquid1With the methanol of 1% volume fraction.
8. a kind of fermentation process of efficiently production recombination human serum albumin according to claim 1, which is characterized in that step
Suddenly in (3) and step (4), the PTM1Preparation method are as follows: take Salzburg vitriol 6g, sodium iodide 0.08g, sulfuric acid monohydrate
Magnesium 3g, two molybdic acid hydrate sodium 0.2g, boric acid 0.02g, cobalt chloride 0.5g, zinc chloride 20g, iron sulfate heptahydrate 65g, biotin
0.2g and sulfuric acid 5mL is dissolved in deionized water together and is settled to 1L.
9. a kind of fermentation process of efficiently production recombination human serum albumin according to claim 1, which is characterized in that step
Suddenly in (4), the helper-inducer agent includes casein hydrolysate, the 1g/L third of the vitamin C of 10mM, volume fraction for 0.5%
Propylhomoserin, 1g/L lysine, 1g/L leucine, 1g/L glutamic acid.
10. a kind of fermentation process of efficiently production recombination human serum albumin according to claim 1, which is characterized in that
In step (5), the centrifugation is centrifuged 5-15min, the temperature of the freezen protective with the revolving speed of 7000-8000rpm at 2-4 DEG C
It is subzero 80 DEG C to subzero 20 DEG C.
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| CN113461804A (en) * | 2021-08-09 | 2021-10-01 | 山东健通生物科技有限公司 | Method for reducing pigment in fermentation process of recombinant human serum albumin |
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