CN100434522C

CN100434522C - Production method of recombination human growth hormone

Info

Publication number: CN100434522C
Application number: CNB2003101087606A
Authority: CN
Inventors: 黄阳滨; 叶建明; 杜碧金; 杨津
Original assignee: Shanghai Newsummit Biopharma Co Ltd
Current assignee: Shanghai Newsummit Biopharma Co Ltd
Priority date: 2003-11-21
Filing date: 2003-11-21
Publication date: 2008-11-19
Anticipated expiration: 2023-11-21
Also published as: CN1618968A

Abstract

The present invention provides an optimized coding sequence of recombinant human growth hormone (rhGH) suitable for yeast expression, a method for producing rhGH with high efficiency and a technology for constructing relevant engineering cells and expressing and purifying rhGH. The optimized growth hormone gene is suitable for yeast expression and has the characteristics of high expression, high stability and high secretion. The present invention can obtain rhGH pure products with high efficiency, convenience and low cost.

Description

Recombinant human somatropin's production method

Technical field

The present invention relates to the genetically engineered field.More specifically, the present invention relates to a kind of High-efficient Production recombinant human somatropin (recombinant human Growth Hormone, method rhGH), and the structure of relevant engineering cell, the expression of rhGH and the technology of purifying.

Background technology

Human growth hormone is human endogenous hormone, by people's Anterior pituitary synthesis secretion, plays an important role aspect growing at human body.The Childhood tethelin shortage can cause children's cretinism (nanism); Pubescence, growth hormone deficiency caused growth retardation; Adult's growth hormone deficiency can cause that overweight, organization unusual (the outer moisture content of adipopexis and born of the same parents descends), bmd descend, lipid constitute worsen, degradation under the insulin sensitivity, with sickness rate such as fracture, cardiovascular disorder, diabetes and motor capacity and mental status descend have certain related.

Human growth hormone can be grown by synthetic bone, internal organ and the whole body of promoting of somatotropin medium (claiming rhIGF-1 again), promotes protein synthesis, accelerates fat and mineral metabolism etc., so play an important role in the human body growth and development process.Human growth hormone only was used for growth hormone deficiency children cretinism originally, come out because of the gene recombination product in the back, clinical consumption is enough to guarantee, so along with going deep into of clinical study, existing indication expands children and pubescence cretinism, adult's growth hormone deficiency, the relevant syndromes etc. of becoming thin of acquired immune deficiency syndrome (AIDS) due to Turner syndrome (gonadal dysfunction syndromes), renal insufficiency and other cause of disease gradually to.

According to up-to-date medical literature report, the countries in the world doctor has carried out deep research at clinical field widely to it according to the mechanism of action of tethelin, the results suggest human growth hormone all has unusual effect, for example recovery of moderate and severe burn, postoperative fatigue syndrome, acute renal failure etc. in a lot of treatment of diseases.These can be considered the potential clinical indication at present.In addition, scientists is also being explored human growth hormone in the purposes aspect anti-ageing healthcare and the beauty treatment, a large amount of experimental results show, it has enhancing body immunologic function and memory, enhance metabolism and body in (particularly belly) fats oxidn, improve bone density, bring high blood pressure down, lessen fatigue, improve sleep or the like effect, be delaying human body caducity, the active drug that retains youth.1996, drugs approved by FDA hGH became unique antiaging hormone medicine.Can estimate that the application on anti-ageing healthcare will become the field that the hGH project has market potential.

217 amino acid of tethelin precursor protein total length, an aminoterminal 1-26 amino acid is a signal peptide.Ripe human growth hormone is a kind of non-glycosylated protein matter of being made up of 191 amino acid, and molecular weight is 22KD.HGH contains two intramolecular disulfide bonds, 4 halfcystines, and these two secondary keys of intramolecularly have played vital role for forming correct ball conformation.Grownup's plasma hGH level is 1-5ng/ml, and the transformation period is 15-20 minute.

Gene engineering expression rhGH starts from 1979, and people such as Goeddel place the hGH gene under the regulation and control of lactose operon, expresses the output that obtains 2.4mg/L in the intestinal bacteria system.Gray and his colleague have realized people rhGH in 1985 secretion expression's (the rhGH gene connects the signal peptide sequence of escherichia coli alkaline phosphatase)---be secreted into colibacillary periplasmic space, but output is too low; After this, Becker utilizes different signal peptide sequences that the secreting, expressing level (being secreted into periplasmic space) of hGH is brought up to about 15mg/L respectively with people such as Chang.1987, people such as Kato were with killer's gene (kill gene) lotus root connection of hGH gene and bacterium, and the secreting, expressing level that obtains hGH reaches 20.5mg/L, and wherein direct secretion reaches 11.2mg/L to the level in the substratum, is trapped in about 8.6mg/L of periplasmic space.People such as Hsiung were in 1989, utilize bacteriocin to discharge the signal peptide of proteic expression and outer membrane protein (outer membrane protein), further improved the expression level of hGH, made its expression level of secreting to the intestinal bacteria substratum bring up to 69.6mg/L.

At present, the rhGH of listing all uses escherichia expression system production, though adopt high density fermentation, expression level is low, accounts for about 10% of tropina, adds the purifying process (needing reversed phase chromatography) of the complexity of bringing thus, the about 30mg/L of productive rate.This low-yield can not be met the need of market.At present, the approval rhGH of FDA Food and Drug Administration (FDA) can be used for the unusual short and small but healthy children of stature, and indication obtains developing, and market capacity further increases, and makes the technical study that promotes industrialization value more meaningful.

Except using escherichia expression system, people also attempt to utilize expression systems such as genus bacillus, cereuisiae fermentum and mammalian cell to produce rhGH.Do not have sugar to intensify the site on the human growth hormone molecule, compare, adopt eukaryotic expression system can not increase sugar chain with prokaryotic system.Regrettably, because factors such as expression level is low, cost height, almost nobody adopts these expression systems to produce rhGH at present.And be wherein most important trend to the research of pichia yeast expression system.

Compare with intestinal bacteria, be fit to the correct folding and modification of expressed human protein molecule in the pichia spp cellular environment more, the product that genetic engineered product all is a solubility, conformation is correct that it is expressed, the hGH of its expression and natural hGH have identical molecular conformation and biological activity; Simultaneously, system compares with intestinal bacteria, the endogenous protein of pichia spp secreting, expressing seldom, the foreign protein of secreting, expressing can reach quite high purity, thereby follow-up purifying process wants much simple.Even and secretion expression's intestinal bacteria system, in the product first product, still contain many intestinal bacteria objectionable constituent (as intracellular toxin), in order thoroughly to remove these compositions, must be through complicated purification step, make that the final yield of product is very low, the highlyest at present also can only accomplish 20-25%.

1997, people such as Olazaran adopted pichia spp (Pichia pastoris) expression system to express rhGH first, but expression level is on the low side, and expression level also has only 13mg/L after optimizing, the meaning that does not have industrialization to produce.

System optimization of the present invention, changed the method for the pichia spp engineering cell that obtains rhGH, and the improvement by zymotechnique, easy purifying process simultaneously obtains high expression level, high yield, has a kind of rhGH production method of industrialization value.

Summary of the invention

Purpose of the present invention just provides a kind of method that efficiently expresses and produce rhGH.

Another object of the present invention provides the expression of corresponding rhGH encoding sequence, carrier, engineering cell and rhGH and the technology of purifying.

In a first aspect of the present invention, a kind of nucleotide sequence of the human growth hormone of encoding is provided, aminoacid sequence shown in the described nucleotide sequence coded SEQ ID NO:2, and nucleotides sequence is shown 95% above homogeny shown in the tethelin coding region of described nucleotide sequence and the SEQ ID NO:1.

In another preference, described nucleotide sequence contains the nucleotide sequence shown in the SEQ ID NO:1.

In a second aspect of the present invention, a kind of expression vector is provided, it contains the above-mentioned nucleotide sequence of the present invention.

In a third aspect of the present invention, a kind of engineering cell is provided, it is to get by homologous recombination behind the above-mentioned sequence transformed host cell of the described expression vector of claim 3 or the present invention, and is integrated with the human growth hormone encoding sequence in the karyomit(e).

In another preference, described engineering cell is pichia spp (Pichia Pastoris).

In a fourth aspect of the present invention, a kind of method of producing human growth hormone is provided, may further comprise the steps:

(a) be fit to cultivate the above-mentioned host cell of the present invention under the expression condition, thus the secreting, expressing human growth hormone; Preferably, described host cell is a pichia spp;

(b) separation and purification goes out the human growth hormone of expression.

In another preference, be integrated with the human growth hormone encoding sequence of 2-30 copy in the genome of described pichia spp host cell.

In another preference, described pichia spp engineering cell comprises and utilizes methanol type fast and utilize methanol type at a slow speed.

In another preference, the culture condition in the step (a) comprises:

Cultivation is divided into cultivation stage and induction period, and cultivation stage is cultivated bacterial concentration and reached OD ₆₀₀Be 40-200, time inductive phase is 24-120hr, and fermentation and inducing temperature remain on 28-30 ℃, micro-PTM ₁Amount is 1-20ml/L, and the pH value of inductive phase is 3-9, and inductive phase, methanol concentration was controlled at 0.5-5%.

In another preference, induce and to add casein hydrolysate (CA), peptone (peptone), milk powder, arginine etc. in the process as protective material.

In another preference, the separation condition of step (b) comprising:

(a) fermented sample is removed thalline by centrifugal and/or filter type, obtains fermentation clear liquid,

(b), or, obtain the pure product that purity reaches (as 95-99.9%) more than 95% directly by anionresin, hydrophobic chromatography method by saltouing and/or after ultrafiltration carries out preliminary purification.

In another preference, sample after 5KD film bag ultrafiltration and concentration displacement damping fluid, Q Sepharose F.F. and phenyl sepharose F.F. chromatography, obtain purity greater than 95%, yield is at the pure product more than 50%.

Description of drawings

Fig. 1 is the structure synoptic diagram of a kind of expression plasmid pPIC9K-rhGH of the present invention.Among the figure, Ampicillin represents ampicillin resistance gene, and Kanamycin represents kalamycin resistance gene.

Fig. 2 is the expression electrophorogram that shakes rhGH under bottle condition of different pH.Each swimming lane is as follows:

1, induce before;

2, protein standard substance (molecular weight is 97.4KD, 66.2KD, 43.0KD, 31.0KD, 20.1KD, 14.4KD from top to bottom)

3,4,5,6,7 be followed successively by and induce sampling in 48 hours under pH7.0,6.0,5.0,4.0 and 3.0 conditions;

Fig. 3 is to the expression electrophorogram before the fermentation technology optimization in the 6.5L fermentor tank.Each swimming lane is as follows:

1. protein standard substance (molecular weight is 97.4KD, 66.2KD, 43.0KD, 31.0KD, 20.1KD, 14.4KD from top to bottom);

2, induce preceding bacterium liquid supernatant;

3-9, be followed successively by the fermented liquid supernatant (4 hours, 8 hours, 16 hours, 24 hours 32 hours, 40 hours and 48 hours) of different inductive phases.

Fig. 4 be in the 6.5L fermentor tank to the expression electrophorogram behind the fermentation technology optimization, can find out that the expression level of rhGH is largely increased after optimizing.Each swimming lane is as follows:

1, protein standard substance (molecular weight is 97.4KD, 66.2KD, 43.0KD, 31.0KD, 20.1KD, 14.4KD from top to bottom);

2, induce preceding bacterium liquid supernatant;

3-8, be followed successively by and induce asynchronous fermented liquid supernatant (8 hours, 16 hours, 24 hours, 32 hours, 40 hours and 44 hours).

Fig. 5 shakes a bottle optimization of material makeup electrophorogram, can find out that use Yeast extract effect is better.Each swimming lane is as follows:

1: shake a bottle supernatant liquor before inducing; 2: blank is induced 48hr; 4:Marker

3,5,6,7: be followed successively by interpolation Yeast extract and induce 24hr and 48hr to shake a bottle supernatant liquor (6 and 7 for repeating)

8,9,10,11: be followed successively by interpolation CA and induce 24hr and 48hr to shake a bottle supernatant liquor (10 and 11 for repeating)

12,13,14,15: be followed successively by interpolation Peptone and induce 24hr and 48hr to shake a bottle supernatant liquor (14 and 15 for repeating).

Embodiment

Extensive studies finds that the low one of the main reasons of expression amount of human growth hormone in the existing production technology is that gene order is without optimization to the inventor by going deep into.The inventor is by the optimization design of gene coded sequence, change the human growth hormone encoding sequence (SEQ ID NO:1) after optimizing over to methyl alcohol and utilize type pichia spp (P.pastoris), thereby realized the high-level secretory expression rhGH of rhGH, finished the present invention on this basis.

In another preference, the present invention further improves the expression level of rhGH significantly also by fermentation technology optimization.Because expression level height, reach more than the 1000mg/L, and be solubility expression, bring very big facility to purifying process, by two-step chromatography technology, increase substantially the product yield, the rhGH product that obtained expression amount height, specific activity height, purify conveniently, yield is high and proterties is stable, reduce production costs greatly, be fit to very much scale operation.

The invention provides a kind of optimization, the human growth hormone encoding sequence that is particularly suitable in yeast cell, expressing.This sequence is designed according to principles such as pichia spp codon-bias.The encoding sequence of the rhGH that designs carries out full gene with ordinary method and synthesizes, or carries out point mutation on the cDNA that obtains by PCR method.

Introduce the specificity restriction enzyme site respectively at 5 of optimized gene ' end and 3 ' end, the ordinary method with molecular cloning is cloned into expression vector (as pPIC9, pPIC9K etc.) with optimized gene.Then, transform, be integrated into the P.pastoris host cell chromosome, utilize ordinary method (as G418 resistance or Southern blotting) to select high copy transformant, shake the bottle expression and select the high expression level engineering cell.Engineering cell can be to utilize methanol type (Mut fast ⁺) or utilize methanol type (Mut at a slow speed ^s).

The copy number that is integrated into the rhGH encoding sequence in the pichia spp karyomit(e) (or genome) is not particularly limited, and can be 1-50 or higher, is generally 2-30.

After obtaining engineering cell, just can be under the condition that is fit to the culturing engineering cell.All substratum that is suitable for the pichia spp growth, expresses all can be used for the present invention.For example, suitable medium comprises (but being not limited to) following substratum:

The YPD flat board	Yeast extract	1％
The YPD flat board	Yeast extract	1％	Peptone	2％
	Glucose	2％	Peptone	2％
	Glucose	2％	Agar	1％
The BMGY nutrient solution	Yeast extract	1％	Agar	1％
The BMGY nutrient solution	Yeast extract	1％	Peptone	2％
	Potassium phosphate buffer, pH 6.0	100mM	Peptone	2％
	Potassium phosphate buffer, pH 6.0	100mM	YNB	1.34％
	Vitamin H	(4×10 ^-5)％	YNB	1.34％
	Vitamin H	(4×10 ^-5)％	Glycerine	1％
The BMMY nutrient solution	Yeast extract	1％	Glycerine	1％
The BMMY nutrient solution	Yeast extract	1％	Peptone	2％
	Potassium phosphate buffer, pH 6.0	100mM	Peptone	2％
	Potassium phosphate buffer, pH 6.0	100mM	YNB	1.34％
	Vitamin H	(4×10 ^-5)％	YNB	1.34％
	Vitamin H	(4×10 ^-5)％	Methyl alcohol	0.5％

A kind of preferred shake-flask culture condition is: the mono-clonal on the picking YPD flat board is a shake-flask culture liquid with BMGY, is cultured to OD ₆₀₀Be 1-40, with BMMY nutrient solution abduction delivering, induce 1 to 7 day after, the rhGH output of nutrient solution supernatant can reach 50-200mg/L.

For scale operation rhGH, need be on fermentor tank the culturing engineering cell, express rhGH, therefore the pilot scale expression condition to the P.pastoris engineering cell is optimized, the fermentation scale is from 1L to 300L, fermentation capacity is linear amplification.Optimize the back expression amount greater than the 1000mg/L fermented liquid.

Through experiment repeatedly of the present invention, expression condition is optimized as follows:

1. for the selection of substratum, can select minimal medium during ferment tank, also can on the minimal medium basis, necessarily change, but contained ion composition should be similar to minimal medium.

The inorganic salt fermention medium	85％H ₃PO ₄	26.7ml/L
The inorganic salt fermention medium	85％H ₃PO ₄	26.7ml/L		CaSO ₄·2H ₂O	1.0g/L
	K ₂SO ₄	18.2g/L		CaSO ₄·2H ₂O	1.0g/L
	K ₂SO ₄	18.2g/L		MgSO ₄	7.27g/L
	KOH (solid)	4.13g/L		MgSO ₄	7.27g/L
	KOH (solid)	4.13g/L		PTM ₁	4.35ml/L
	Glycerine			PTM ₁	4.35ml/L
	Glycerine		4％(w/v)
PTM ₁	CuSO ₄		4％(w/v)	6g/L
PTM ₁	CuSO ₄		KI	6g/L	0.08g/L
	MnSO ₄.H ₂O		KI	3g/L	0.08g/L
	MnSO ₄.H ₂O		Two molybdic acid hydrate sodium	3g/L	0.2g/L
	Boric acid		Two molybdic acid hydrate sodium	0.02g/L	0.2g/L
	Boric acid		ZnCl ₂	0.02g/L	20g/L
	Cobalt chloride		ZnCl ₂	0.5g/L	20g/L
	Cobalt chloride		FeSO ₄.7H ₂O	0.5g/L	65g/L
	Vitamin H		FeSO ₄.7H ₂O	0.2g/L	65g/L
	Vitamin H		H ₂SO ₄	0.2g/L	5ml/L

2. with regard to temperature controlling, the fermentation of rhGH and inducing temperature remain on 28-30 ℃.

3. with regard to the pH value of inductive phase, inductive phase, pH was controlled at 3-9;

4. with regard to the control of dissolved oxygen (DO), DO is controlled at 20-90%.Keeping of dissolved oxygen can solve with the feeding of oxygen/air mixed gas.

5. with regard to the stream of feed supplement added, the feed supplement kind should comprise carbon sources such as glycerine, methyl alcohol, glucose, separately feed supplement or mix feed supplement.

6. with regard to PTM ₁The amount of (mixed trace elements): initial cultivation stage PTM ₁Amount be 1-12ml/L training liquid, be the 2-20ml/L feed supplement in feed supplement stage add-on.

7. with regard to the inductive phase methanol concentration, conventional induced concentration all can be used for the present invention, and methanol concentration is controlled at 0.5-5% usually.

8. protective material: can be casein hydrolysate (CA), peptone (peptone), milk powder etc., concentration 0.1-10mg/mL; Or 0.1-1M arginine.

9. with regard to induction time, being not particularly limited, being generally 12-160 hour, preferably is 24-120 hour.

The rhGH that expresses is present in training liquid supernatant, and expression level accounts for the fermented liquid supernatant total protein more than 50%, and the purifying complex degree is descended greatly.Usually, fermented sample is earlier removed cell in modes such as centrifugal, filtrations, and fermentation clear liquid contains target protein matter rhGH.Fermentation clear liquid can by saltout, method such as ultrafiltration carries out carrying out chromatography purification again behind the preliminary purification, also can directly carry out chromatography purification.

Chromatographic technique comprises technology such as cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, hydrophobic chromatography, affinity chromatography.Through different chromatographic techniques, chromatography process relatively, the chromatography method of optimization comprises:

1. anion-exchange chromatography:

The anion-exchange chromatography medium includes, but is not limited to Q-Sepharose, DEAE-Sepharose.If the salt concn of fermented sample is higher, influence combines with Ion Exchange Medium, then needs to reduce salt concn before carrying out ion exchange chromatography.Sample can carry out the replacing of level pad with means such as dilution, ultrafiltration, dialysis, gel permeation chromatographies, until with corresponding ion exchange column balance liquid system similarity, go up sample then, carry out the gradient elution of salt concn or pH.

2. hydrophobic chromatography:

Hydrophobic chromatoghaphy medium includes, but is not limited to Phenyl-Sepharose, Butyl-Sepharose, Octyle-Sepharose.Sample is by adding NaCl, (NH ₄) ₂SO ₄Improve salt concn etc. mode, go up sample then, by reducing salt concn method wash-out.Remove hydrophobicity by hydrophobic chromatography foreign protein than big-difference is arranged.

3. gel permeation chromatography

Hydrophobic chromatoghaphy medium includes, but is not limited to Sephacryl, Superdex, Sephadex class.By gel permeation chromatography exchange buffering system, or further consummate.

Can obtain the pure product of rhGH through above-mentioned purifying, the purifying yield is generally more than 40%, and purity is more than 95%, and the about 500mg/L of pure product yield trains liquid.And at present sophisticated by escherichia coli expression, purifying rhGH, the about 30mg/L of its pure product yield, as seen, the present invention will promote the industrialization value of rhGH greatly.

HGH behind the purifying can make various formulations with ordinary method, as lyophilized injectable powder.Select certain stablizer, also can add protective materials such as tensio-active agent, antioxidant simultaneously, and suitable damping fluid carries out lyophilize.The goods that obtain have that loss of activity is little, moisture content is low, good stability, be easy to characteristics such as prolonged preservation.

RhGH also can make aqueous injection, sprays, microsphere sustained-release agent, and makes prolonged action preparation etc. through the PEG modification.

In an example of the present invention, made up the P.pastoris yeast expression engineering cell of rhGH, methanol induction, high-level secretory expression rhGH does not need renaturation.

In another example of the present invention, parameter optimization by fermentation improves the expression level of rhGH.

Because of expressing protein belongs to the exocytosis type, the fermentation supernatant can directly carry out purifying.In another example of the present invention, through purifying, obtain the pure product of rhGH, the purifying yield is more than 50%, and purity is more than 95%, and the about 500mg/L of pure product yield trains liquid.

In another example of the present invention, stoste adds suitable auxiliary material behind the purifying, makes the powder ampoule agent for injection of rhGH.Stability test shows that this powder injection is stable.

Major advantage of the present invention is:

(1) growth hormone gene after the optimization is fit to yeast expression very much, has high expression level, high stable, high excretory characteristics.

(2) the host P.pastoris that contains the growth hormone gene of optimization has the characteristic of high-density growth, is very suitable for the fermentative production of genetically engineered drug large scale and high density.The hGH that P.pastoris expresses directly is secreted into outside the born of the same parents with solubility, tool activity form; Product must renaturation, can directly carry out purifying; Owing to the expression level height, the purifying process complexity is simplified greatly simultaneously, and yield is greatly improved.

(3) compare with the prokaryotic system expression, industrialization value is improved significantly.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning experiment guide (New York:ColdSpring Harbor Laboratory Press, 1989); People such as Jan-Christer Janson, Proteinpurification (John Wiley ﹠amp; Sonss, Inc., 1998) described in condition, or the condition of advising according to manufacturer.

The structure of embodiment 1.hGH pichia yeast expression system

1. the acquisition of goal gene

According to the aminoacid sequence of natural human tethelin, press principles such as pichia spp codon-bias, purpose of design gene and full gene are synthetic, the dna fragmentation of its sequence shown in SEQ ID NO:1.Introduce XhoI, EcoRI restriction enzyme site respectively at its 5 ' end and 3 ' end simultaneously.

Obtain human growth hormone gene (SEQ ID NO:1) with total synthesis method, this genes encoding human growth hormone aminoacid sequence is shown in SEQ ID NO:2.

2. the acquisition of the structure of expression plasmid pPIC9K/rhGH and high expression level engineering cell

Building process as shown in Figure 1.The full gene rhGH of synthetic is cloned into earlier among the pPIC9 (Invitrogen company) with XhoI+EcoRI partially digested (an XhoI site is arranged in the middle of the Yin Jiyin), obtains pPIC9/rhGH, and order-checking conclusive evidence dna sequence dna is correct.Then, pPIC9K plasmid (Invitrogen company) with as reclaim big fragment behind the Bpu1102I+EcoRI enzymolysis, the small segment of the pPIC9/rhGH recovery of cutting with same enzyme is connected and spends the night transformed into escherichia coli DH5 α, the screening transformant, the pPIC9K plasmid of rhGH gene is inserted in acquisition

The recombinant plasmid pPIC9K/rhGH linearizing that builds, electroporation transformed host cell P.pastoris, coating MD flat board, on the YPD flat board of different concns G418 (as 1,2,3,4,5mg/L), screen then, obtain a collection of high resistance engineering strain, expression screening obtains a plurality of strains that efficiently express in test tube, induces and expresses the about 50-200mg/L training of output liquid (accompanying drawing 2) after 48 hours.

Embodiment 2pH is to the influence of expression level

Medium pH may influence the level that the pichia spp engineering cell is expressed rhGH.Get mono-clonal, be inoculated in the BMGY primary seed solution, cultivate 17-20hr; , cultivate about 24hr in the 500ml of 50ml substratum Erlenmeyer flask in 1: 10 ratio two-stage inoculation, 1% methanol induction, the pH of induction period need according to the form below to regulate, and SDS-PAGE detects expression.

Experiment numbers	Experiment condition
Experiment numbers	Experiment condition		1	PH3.0
2	PH4.0		1	PH3.0
2	PH4.0	3	PH5.0
4	PH6.0	3	PH5.0
4	PH6.0	5	PH7.0

Experimental result shows, expresses rhGH and express better (accompanying drawing 3) at pH at 3.0-4.0 in shaking bottle.

Embodiment 3 protective materials are to the influence of expression level

Protective material can prevent the degraded of target protein.Get mono-clonal, be inoculated in the BMGY primary seed solution, cultivate 17-20hr; , in the 500ml of 50ml substratum Erlenmeyer flask, cultivate about 24hr in 1: 10 ratio two-stage inoculation, 1% methanol induction, according to the form below adds different protective materials, and SDS-PAGE detects expression.

Experimental result shows that Yeast extract effect is better in three kinds of protective materials, and 1% concentration is also than 0.5% concentration good (accompanying drawing 4).

Embodiment 4.PTM ₁Amount is to the influence of expression level

On the basis of embodiment 2, following two kinds of methods:

Method one:

PTM in the basic medium ₁Amount: 2ml/L

PTM in the induction period feed supplement ₁Amount: 2ml/L feed supplement

Method two:

PTM in the basic medium ₁Amount: 2ml/L

PTM in the induction period feed supplement ₁Amount: 10ml/L feed supplement

The result is as shown in the table, shows when the induction period feed supplement, and the amount of PTMx is (3-20ml/L feed supplement) well with higher.

	Method one	Method two
	Method one	Method two	The rhGH expression amount	120mg/L	180mg/L

Embodiment 5. pilot scale fermentation conditions are selected and are optimized

NBS BioFlo 3000 fermentor tanks (6.5L)

Through after the above-mentioned optimization, the rhGH expression amount accounts for fermented supernatant fluid total protein 60%, and expression amount reaches 1500mg/L above (accompanying drawing 4 and Fig. 5).

Embodiment 6.rhGH purifying

Centrifugal 10 minutes of 4 ℃ of following 5000rpm of the fermented liquid of embodiment 4 get supernatant.

Use the Millipore ultra-fine filter, the ultra-filtration membrane molecular weight that dams is 5KD, leaves and takes phegma during ultrafiltration.Fermented liquid supernatant ultrafiltration to volume is left about 500ml, and (PB pH7.4), continues ultrafiltration with the 10mM phosphate buffered saline buffer in adding; This program repeatedly flushes closely until the conductivity water of the gentle 10mM PB of the conductivity water of sample (pH7.4) damping fluid.

Chromatography 1:

Chromatography media: Q Sepharose F.F. (Phamarcia)

Damping fluid: solution A: 10mM PB (pH 7.4)

Solution B: 10mM PB+1M NaCl (pH 7.4)

Last sample: the rhGH solution (about 1L, electricity is led to about 4000us/cm) of ultrafiltration and concentration is gone up sample

Clean: (column volume) solution A is cleaned chromatography column down together with 6CV after going up sample

Gradient: clean the back directly with 30% solution B wash-out target protein

Collect: collect the albumen under the 30%B wash-out

Chromatography 2:

Chromatography media: phenyl-sepharose (Phamarcia)

Damping fluid: solution A: 10mM PB (pH 7.4)

Solution C: 10mM PB+3M NaCl (pH 7.4)

Last sample: the sample that chromatography 1 is obtained adds solid NaCl to 3.0M, last sample

Gradient: 100%A directly cleans

Collect: collect the target protein that 100%A washes

Sample is through this 2 step chromatography, and promptly after anion-exchange chromatography, the hydrophobic chromatography, purity is increased to 95%, yield is at the pure product of the rhGH 50% or more, and pure product yield 500mg/L trains the liquid supernatant.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Shanghai Xinshengyuan Medicine Research Co., Ltd.

＜120〉recombinant human somatropin's production method

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＜223〉the human growth hormone encoding sequence of You Huaing

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catcagctag cctttgacac ctaccaggag tttgaagagg cctatatccc caaggaacag 120

aagtattcat tcctgcagaa cccccagacc tccctctgtt tctcagaatc gattccgaca 180

ccctccaatc gcgaggaaac acaacagaaa tccaacctag agctcctccg cataagcttg 240

ctgctcatcc agtcgtggct cgagcccgtg cagttcctga ggagtgtctt cgccaacagc 300

ctggtctacg gcgcctctga ttcgaacgtg tacgacctgc tgaaggacct agaggaaggg 360

atccaaacgc tgatggggag gctggaagat ggcagcccgc ggactgggca gatcttcaag 420

cagacctaca gcaagttcga cacaaactca cacaacgatg acgcactact caagaactac 480

gggctgctct actgcttcag gaaggacatg gacaaggtcg agacattcct gcgcatcgtg 540

cagtgccgct ctgtggaggg cagctgtggc ttctag 576

<210>2

<211>191

<212>PRT

＜213〉homo sapiens (Homo sapiens)

<400>2

Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg

1 5 10 15

Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu

20 25 30

Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro

35 40 45

Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg

50 55 60

Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu

65 70 75 80

Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val

85 90 95

Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp

100 105 110

Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu

115 120 125

Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser

130 135 140

Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr

145 150 155 160

Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe

165 170 175

Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe

180 185 190

Claims

1. the nucleotide sequence of the human growth hormone of encoding is characterized in that, the aminoacid sequence shown in the described nucleotide sequence coded SEQ ID NO:2, and also the tethelin coding region of described nucleotide sequence is and nucleotide sequence shown in the SEQ ID NO:1.

2. an expression vector is characterized in that, it contains the described nucleotide sequence of claim 1.

3. an engineering cell is characterized in that, it by described expression vector of claim 2 or the described sequence transformed host cell of claim 1 after homologous recombination get, and be integrated with the human growth hormone encoding sequence in the karyomit(e).

4. engineering cell as claimed in claim 3 is characterized in that, it is pichia spp (PichiaPastoris).

5. a method of producing human growth hormone is characterized in that, may further comprise the steps:

(a) be fit to cultivate engineering cell as claimed in claim 4 under the expression condition, thus the secreting, expressing human growth hormone, and described engineering cell is a pichia spp;

6. method as claimed in claim 5 is characterized in that, is integrated with the human growth hormone encoding sequence of 2-30 copy in the genome of described pichia spp engineering cell.

7. method as claimed in claim 5 is characterized in that, described pichia spp engineering cell comprises and utilizes methanol type fast and utilize methanol type at a slow speed.

8. method as claimed in claim 5 is characterized in that, the culture condition in the step (a) comprises:

9. method as claimed in claim 5 is characterized in that, the separation condition of step (b) comprising:

(b) by saltouing and/or after ultrafiltration carries out preliminary purification, or directly by anionresin, hydrophobic chromatography method, thereby the pure product that purity reaches 95-99.9% obtained.