A kind of extracting method of insulin Glargine precursor protein
This application claims and submit Patent Office of the People's Republic of China, Application No. 201210475070.3, invention on November 21st, 2012
The priority of the Chinese patent application of entitled " extracting method of a kind of insulin Glargine precursor protein ", entire contents is passed through
Quote and be incorporated in the present application.
Technical field
The present invention relates to biochemical field, particularly relate to the extraction side of a kind of insulin Glargine precursor protein
Method.
Background technology
In treating diabetes, insulin to control blood glucose fluctuation, complication prevention has irreplaceable effect, is mesh
One of front maximally efficient treating diabetes means.In recent years, along with the development of insulin technology, have developed insulin type seemingly
Thing.One of which Recent Development of Long-acting Insulin Analogs-insulin Glargine, is accepted by increasing doctor and patient and uses.
Insulin Glargine is a kind of biosynthetic human insulin analog utilizing recombinant DNA technology to produce.It is people
Insulin B chain carboxyl terminal adds two arginine, also the agedoite of A chain carboxyl terminal A21 position is replaced to simultaneously
Glycine, after subcutaneous injection, the sustainable slow release insulin Glargine of microscopic precipitate formed because acid solution is neutralized, continues
Absorbed into serum also slow, thus produce up to 24 hours steadily without the foreseeable blood drug level of peak value.Therefore, timing every day skin
Insulin Glargine of hemostasis, can meet the human body needs to basal insulin, also mitigates patient and wants multiple injection pain
Bitter.In the case of persistently improving glycemic control, insulin Glargine can reduce hypoglycemia incidence rate.
The method preparing insulin Glargine is typically to utilize technique for gene engineering to be connected to by insulin Glargine precursor-gene
On expression vector, then it is transformed into microbial bacteria expression system and carries out fermenting and producing insulin Glargine precursor protein, then thalline
Separation and Extraction precursor protein enzyme action process and obtain insulin Glargine.The most common Microbial Expression Systems has escherichia coli table
Reach system and yeast expression system.Escherichia expression system product mostly is inclusion body, by contrast, yeast expression system without
Loaded down with trivial details and the high consumption operating procedure of inclusion body refolding strategy, and yeast nutritional requirement is low, growth fast, culture medium is cheap, table
The foreign protein reached can be secreted into outside born of the same parents, and intrinsic protein secretion is less, beneficially the separation of foreign protein and purification.
Extracting insulin Glargine precursor protein is to produce the indispensable step of insulin Glargine, and present stage extracts sweet essence pancreas
Island element precursor protein is usually fermentation liquid is the most preprocessed, and direct collected after centrifugation supernatant, thalline goes out of use.But due to
Destination protein character is different, has the destination protein of part to be likely to be present in discarded thalline, the discarded mesh that will cause acquisition
Product amount be substantially reduced.Therefore, specific destination protein fermentation liquid is carried out pretreatment, is conducive to improving yield.Fermentation liquid
Pretreatment mode has temperature regulation, pH regulator, flocculates and cohesion etc., but is mostly all used for antibiotic pretreatment, and main purpose is
Improve filter effect etc..As a example by regulation pH value, after the purpose of existing report such as US6218385 fermentating liquid acidification is inactivation fermentation
Remaining microorganism, prevents the broth viscosities that meat soup self-dissolving is relatively low with maintenance;CN200610011694.4 fermentating liquid acidification
Purpose is to process the convenient filtration of pod membrane of antibacterial;The purpose of CN200710026682.3 fermentating liquid acidification be make destination protein not by
Activated carbon adsorption;The purpose of CN200810233835.6 fermentating liquid acidification is to prevent glutathion oxidized;
CN200910222792.6 is to precipitate dilution acid adding auxiliary dissolving natamycin after fermentation is centrifuged, but in above-mentioned report the most not
Relate to insulin Glargine precursor protein fermentation liquid, and the starting point of its acid adding is not to increase percent protein.People's islets of langerhans simultaneously
After the element the most acidified process of Precurosor fermentation liquid, the response rate of its precursor protein does not significantly improve.Additionally, other
The report of pretreated fermentation liquid is also not directed to the raising of insulin Glargine precursor protein content.
It can thus be seen that about how to improve insulin Glargine precursor protein content during extracting in prior art
Report almost without.Therefore, need to provide one can increase insulin Glargine precursor protein to contain in insulin production field
The extracting method of amount, thus improve the production efficiency of insulin Glargine.
Summary of the invention
In view of this, it is an object of the invention to provide the extracting method of a kind of insulin Glargine precursor protein so that should
Extracting method can improve the content of insulin Glargine precursor protein.
To achieve these goals, the present invention provides following technical scheme:
The extracting method of a kind of insulin Glargine precursor protein, regulates pH by insulin Glargine precursor protein fermentating liquid acidification
Value is 1-4, and then centrifuging and taking supernatant i.e. obtains insulin Glargine precursor protein.
Present invention applicant extracts the defect of insulin Glargine precursor protein method for existing direct centrifugal segregation thalline,
Through further investigation, the pH value with acid regulation insulin Glargine precursor protein fermentation liquid is 1-4 before centrifugation so that in fermentation liquid
Insulin Glargine precursor protein content is improved, and its precursor protein content is at most increased up to 83%.
Wherein, as preferably, described acid is hydrochloric acid, glacial acetic acid, sulphuric acid or phosphoric acid, more preferably in hydrochloric acid, phosphoric acid
Plant or two or more;Described pH value is preferably adjusted to 1-3, is more preferably adjusted to 2.
Insulin Glargine precursor protein typically by insulin human B chain (end increase by two arginine)+connection peptides (i.e. C peptide,
Insulin Glargine precursor protein can be without C peptide)+insulin human A chain (A21 position is glycine) composition, wherein said A chain and B
Chain amino acid sequence is known in the art, fixes, and C peptide sequence is not fixed, and there is also the situation without C peptide simultaneously.The present invention is
The described extracting method of checking can be applicable to the insulin Glargine precursor protein fermentation liquid of different C peptide sequence, chooses this area 5
Kind of different C peptide sequences are as connection peptides and do not use connection peptides (i.e. the glycine of B chain end arginine and A start of chain is direct
It is connected by amido link) to extract, result shows, and without compared with adding the extracting method of acid for adjusting pH value, of the present invention
Extracting method all can improve the content of insulin Glargine precursor protein.
As preferably, the C peptide sequence of described insulin Glargine precursor protein be aminoacid sequence shown in SEQ ID NO:1,
Aminoacid sequence shown in aminoacid sequence, SEQ ID NO:4 shown in aminoacid sequence shown in SEQ ID NO:2, SEQ ID NO:3
Or aminoacid sequence shown in SEQ ID NO:5.
Insulin Glargine precursor protein fermentation liquid of the present invention can be by genetic engineering routine techniques by insulin Glargine
Precursor-gene carrier construction imports in microbial strains, then fermentation culture and get final product, and those skilled in the art can be according to microorganism
Bacterial strain rationally determines that fermentation culture operates and obtains fermentation liquid, and this can realize easily.And technical solution of the present invention exists
The pretreated fermentation liquid in how, does not limit the acquisition pattern of fermentation liquid, and existing fermentation culture method is at the gene one imported
On the premise of cause, the insulin Glargine precursor protein obtained is the most identical, and difference is the difference of fermentation efficiency.
As preferably, the present invention uses insulin Glargine precursor protein yeast expression fermentation liquid or insulin Glargine precursor egg
White escherichia coli expression fermentation liquid.
As preferably, described yeast is Pichia sp. or saccharomyces cerevisiae.
As preferably, described insulin Glargine precursor protein yeast expression fermentation liquid is by importing insulin Glargine precursor base
The culture propagation of cause cultivates and utilizes glycerol and methanol secreting, expressing to obtain.Wherein, during the fermentation, different nitrogen sources is used
Cultivate for cell with carbon source, belong to this area conventional method.In like manner, glycerol and methanol are carbon source conventional in fermentation culture,
Can also be replaced with similar substance, such as glucose and methylamine, this is that those skilled in the art are capable of.
Above-mentioned fermentation culture and before utilizing glycerol and methanol induction yeast and E. coli secretion to express insulin Glargine
Body protein belongs to this area conventional method, illustrates as a example by yeast:
Fermentation medium is by BSM, and 1% yeast extract (Yeast Extract) and PTM1 form.BSM and yeast extract
After 121 DEG C of high temperature sterilize 30m, with 50% ammonia pH is adjusted to 5.0, then adds through the PTM1 of aseptic filtration by 4mL/L and get final product.
Seed liquor is cultivated: draw YPD flat board with the strain preserved, and chooses lawn inoculation containing 50mLYPG after 30 DEG C of incubation 24h
250mL shaking flask, 30 DEG C, 250rpm, cultivate 24h as first order seed.Two of 500mL YPG are contained with primary seed solution inoculation
1L shaking flask, 30 DEG C of 250rpm, cultivate 15-20h, as secondary seed.1L secondary seed solution is injected containing 9L fermentation medium
In 30L fermentation tank.Fermentation initial parameters is set to: temperature 30 DEG C, pH5.0, dissolved oxygen 100%, rotating speed 300rpm, ventilation 10L/
min。
Fermentation is divided into three steps: the first step, fermentation medium cultivate about 20h;Second step, add with the speed stream of 10mL/h/L and contain
50% glycerol (final concentration of 0.5-0.9%) of 12mL/L PTM1,1-3h.3rd step, add containing 12mL/ with the speed stream of 6mL/h/L
The methanol (final concentration of 0.5-1.0%) of L PTM1 is induced.For making the DO value in Induction Process be not less than 25%, first need to be regulated
Alcohol stream rate of acceleration, induces 72h, and bacterium solution OD600 is lower tank after reaching more than 500.
Choose the fermentation liquid after different yeast expression system ferments, then according to extracting method of the present invention extracts
Insulin Glargine precursor protein, compared with the extracting method regulated without acid adding, hence it is evident that improves insulin Glargine precursor protein and contains
Amount.The Escherichia coli fermentation simultaneously utilizing recombination cultivates the insulin Glargine precursor protein fermentation liquid obtained, and acid adding regulates
PH value has also reached to improve the effect of precursor protein content.
From above technical scheme, the present invention regulated insulin Glargine precursor by acid adding before centrifugation thalline
Protein fermentation liquor pH value is 1-4 so that the insulin Glargine precursor protein content after extraction is improved, and compensate for the most centrifugal
Remove the defect that thalline causes destination protein to lose, improve insulin Glargine production efficiency.
Detailed description of the invention
The embodiment of the invention discloses the extracting method of a kind of insulin Glargine precursor protein.Those skilled in the art are permissible
Use for reference present disclosure, be suitably modified technological parameter and realize.Special needs to be pointed out is, all similar replacements and change are to ability
Being apparent from for field technique personnel, they are considered as being included in the present invention.The method of the present invention has been passed through preferably
Embodiment is described, and related personnel substantially can be to side as herein described in without departing from present invention, spirit and scope
Method is modified or suitably changes and combine, and realizes and applies the technology of the present invention.
In order to be further appreciated by the present invention, it is described in detail below in conjunction with embodiment.
Embodiment 1:
Microbial strains: Pichia pastoris GS115 (commercially available)
C peptide sequence: aminoacid sequence shown in SEQ ID NO:1
Fermentation liquid: obtained by the Pichia pastoris GS115 fermentation culture importing insulin Glargine precursor-gene.
Surveying fermentation liquid pH value is 5.0, and being then centrifuged for taking supernatant HPLC and surveying the concentration of its insulin Glargine precursor is standard
Value 100, as comparison, then takes the fermentation liquid of 16 parts of same volumes, use 1.2mol/L hydrochloric acid, glacial acetic acid, 10% concentrated sulphuric acid and
95% phosphoric acid adjusts pH to be 1,2,3,4 respectively, is then centrifuged for taking supernatant detection and contrasts, the results are shown in Table 1.
The different acid for adjusting pH value insulin Glargine precursor HPLC testing result of table 1
pH |
1 |
2 |
3 |
4 |
5(compares) |
1.2mol/L hydrochloric acid |
173 |
179 |
171 |
129 |
100 |
Glacial acetic acid |
13.3 |
64.6 |
141 |
135 |
100 |
10% sulphuric acid |
124 |
154 |
145 |
119 |
100 |
95% phosphoric acid |
141 |
183 |
158 |
129 |
100 |
As shown in Table 1, adding acid treatment according to the inventive method, when pH value is 3 and 4, all acid all improve sweet essence pancreas
The concentration of island element precursor, and when pH value is 1 and 2, outside deicing acetic acid, its spent acid all improves the dense of insulin Glargine precursor
Degree.Owing to glacial acetic acid acidity is more weak, regulation solution ph is 2 be diluted to original volume 2.5 times, and regulation solution ph is 1 dilution
To 12.6 times of original volume, so significantly diluting concentration, but what precursor total amount still increased under the conditions of comparing pH3-5.From whole
Body result is seen, the concentration of pH insulin Glargine precursor between 1 and 3 improves higher, thus can deduce the method for the invention energy
Enough significantly improve the content of insulin Glargine precursor protein.
Embodiment 2:
Microbial strains: Pichia pastoris GS115 (commercially available)
C peptide sequence: aminoacid sequence shown in SEQ ID NO:1
Fermentation liquid: with embodiment 1
Surveying fermentation liquid pH value is 5.0, and being then centrifuged for taking supernatant HPLC and surveying the concentration of its insulin Glargine precursor is standard
Value 100, as comparison, then takes the fermentation liquid of 10 parts of same volumes, uses 1.2mol/L hydrochloric acid and 95% phosphoric acid to adjust the pH to be respectively
1.2,1.5,1.8,2.1,2.4, it is then centrifuged for taking supernatant detection and contrasts, the results are shown in Table 2.
The different acid for adjusting pH value insulin Glargine precursor HPLC testing result of table 2
pH |
1.2 |
1.5 |
1.8 |
2.1 |
2.4 |
5(compares) |
1.2mol/L hydrochloric acid |
170 |
172 |
172 |
179 |
165 |
100 |
95% phosphoric acid |
133 |
137 |
160 |
185 |
178 |
100 |
As shown in Table 2, add acid treatment according to the inventive method, when pH value is 1.2,1.5,1.8,2.1,2.4, all acid
All significantly improve the concentration of insulin Glargine precursor, thus can deduce that the method for the invention can significantly improve sweet essence pancreas
The content of island element precursor protein.
Embodiment 3:
Microbial strains: saccharomyces cerevisiae MT663(is commercially available)
C peptide sequence: aminoacid sequence shown in SEQ ID NO:1
Fermentation liquid: obtained by the saccharomyces cerevisiae MT663 fermentation culture importing insulin Glargine precursor-gene.
Surveying fermentation liquid pH value is 5.2, and being then centrifuged for taking supernatant HPLC and surveying the concentration of its insulin Glargine precursor is standard
Value 100, as comparison, then takes the fermentation liquid of 10 parts of same volumes, uses 1.2mol/L hydrochloric acid and 95% phosphoric acid to adjust the pH to be respectively
1.2,1.5,1.8,2.1,2.4, it is then centrifuged for taking supernatant detection and contrasts, the results are shown in Table 3.
The different acid for adjusting pH value insulin Glargine precursor HPLC testing result of table 3
pH |
1 |
2 |
3 |
4 |
5 |
5.2(compares) |
1.2mol/L hydrochloric acid |
161 |
170 |
160 |
119 |
107 |
100 |
95% phosphoric acid |
130 |
174 |
150 |
120 |
106 |
100 |
As shown in Table 3, adding acid treatment according to the inventive method, when pH value is 1,2,3,4, all acid all significantly improve
The concentration of insulin Glargine precursor, thus can deduce that the method for the invention can significantly improve insulin Glargine precursor protein
Content.
Embodiment 4:
In order to verify that extracting method of the present invention is only applicable to the extraction of insulin Glargine precursor protein further, according to
The conventional means of prokaryotic expression is respectively by protaminase precursor-gene, trypsin precursor-gene, Porcine insulin gene, door
Winter insulin precursor-gene and insulin Glargine precursor-gene import fermentation culture secreting, expressing in Pichia pastoris GS115, so
Regulating pH value by interpolation concentrated hydrochloric acid/sodium hydroxide afterwards, after the same terms is centrifugal, the supernatant HPLC to each pH value surveys it
Precursor concentration.Protaminase precursor, Porcine insulin, insulin aspart precursor and insulin Glargine precursor are used under pH5
The concentration that HPLC records is that standard value is set to 100, and the concentration using the HPLC under pH4 to record trypsin precursor is standard value
It is set to 100, the results are shown in Table 4
Table 4 different enzyme, the protein content testing result of insulin precurosor
pH |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
Protaminase precursor |
17 |
54 |
63 |
95 |
100 |
113 |
120 |
Trypsin precursor |
97 |
98 |
99 |
100 |
93 |
67 |
55 |
Porcine insulin |
99 |
96 |
97 |
98 |
100 |
102 |
101 |
Insulin aspart precursor |
97 |
99 |
98 |
97 |
100 |
99 |
98 |
Insulin Glargine precursor |
173 |
179 |
171 |
129 |
100 |
61 |
20 |
By table 4, above-mentioned four kinds of albumen are Preliminary fermentation value pH value is 5-6 when, by acid adding, alkali, only
Insulin Glargine precursor can have a distinct increment in the case of pH reduces, and when pH value is 1-2, in supernatant, precursor concentration is the highest.By
This is it is inferred that extracting method of the present invention is only applicable to the extraction of insulin Glargine precursor protein.
Embodiment 5:
With reference to the method for embodiment 1, the insulin Glargine Precurosor fermentation liquid selecting different C peptide sequence to connect extracts,
Wherein, first group is aminoacid sequence shown in SEQ ID NO:1 without C peptide, second group of C peptide sequence, and the 3rd group of C peptide sequence is SEQ
Aminoacid sequence shown in ID NO:2, the 4th group of C peptide sequence is aminoacid sequence shown in SEQ ID NO:3, the 5th group of C peptide sequence
For aminoacid sequence shown in SEQ ID NO:4, the 6th group of C peptide sequence is aminoacid sequence shown in SEQ ID NO:5, uses pH value
Be the precursor concentration that the HPLC under 5 records be that standard value is set to 100, the results are shown in Table 5.
The insulin Glargine precursor HPLC testing result of the different C peptide sequence of table 5
C peptide sequence |
pH=1 |
pH=2 |
pH=3 |
pH=4 |
pH=5 |
Group 1 |
154 |
170 |
151 |
116 |
100 |
Group 2 |
160 |
168 |
142 |
110 |
100 |
Group 3 |
154 |
166 |
148 |
109 |
100 |
Group 4 |
168 |
172 |
150 |
119 |
100 |
Group 5 |
169 |
179 |
148 |
122 |
100 |
Group 6 |
173 |
180 |
153 |
120 |
100 |
As shown in Table 5, insulin Glargine precursor uses multiple C peptide sequence to connect, in the environment of pH value is 1-4, centrifugal
The concentration of rear supernatant sweet essence precursor reduces with pH value and increases, and pH value be 1 be 2 with pH value time concentration higher, show
Invent described extracting method and can be applicable to all insulin Glargine precursor protein fermentation liquids, and its protein content can be improved.
The explanation of above example is only intended to help to understand method and the core concept thereof of the present invention.It is right to it should be pointed out that,
For those skilled in the art, under the premise without departing from the principles of the invention, it is also possible to the present invention is carried out
Some improvement and modification, these improve and modify in the protection domain also falling into the claims in the present invention.