JPH0759598B2 - Method for producing human insulin-like growth factor I - Google Patents

Description

Detailed Description of the Invention [Field of Industrial Application] This invention relates to a novel method for producing human insulin-like growth factor I (hereinafter referred to as IGF-I), which is useful as a medicine.

[Prior Art] IGF-I is a known compound, and its primary structure has been reported by RE Humbel et al. to be as follows: [T. L. Brundel, S. Bedarkar, E. Lindelknecht and R. E. Humbel: Proceedings of the National Academy of Sciences of the USA,
75, pp. 180-184 (1978) (TL Blunde
ll, S. Bedarkar, E. Rinderknecht and REHumbel: Proc.N.
atl. Acad. Sci. USA, 75 , 180-184 (1978)), and E. Lindelknecht and R. E. Hambel: Journal of Biological Chemistry, Vol. 25
3, No. 2769 (1978) (E. Rinderknecht and R.
E. Humbel: J. Biol. Chem. 253 , 2769 (1978)] (Wherein, A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is
is glycine, I is isoleucine, K is lysine, L is leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T
(V stands for threonine, V stands for valine, Y stands for tyrosine, and the three solid lines connecting C and C stand for -S-S bonds.) Hambel et al. isolated IG with the above primary structure from human blood.
The inventors of the present invention isolated IGF-I and determined its structure, and the inventors of the present invention isolated IGF-I obtained by recombinant gene technology with the amino acid sequence: (In the formula, A, C, D, E, F, G, I, K, L, M,
N, P, Q, R, S, T, V and Y have the same meanings as above), and the resulting reduced IGF-I
Oxidized IGF-I was obtained by oxidizing IGF-I. In other words, reduced IGF-I is a product of the cysteine bonds between three pairs of cysteines in IGF-I being substituted.
The S-S-bond is broken to form linear IGF-I, and this reduced IGF-I is oxidized to form oxidized IGF-I.
IGF-I, i.e., IGF-I having three pairs of -S-S- bonds
In the course of leading to this discovery, the present inventors discovered an IGF-I having a primary structure different from that of Hambel et al.

Its primary structure is of the formula (In the formula, A, C, D, E, F, G, I, K, L, M,
The letters N, P, Q, R, S, T, V, and Y as well as the three solid lines connecting C and C have the same meanings as before.) and its biological properties were found to be different from those isolated by Hambel et al.

In this specification, IGF-I having the primary structure represented by formula (I-a) isolated by Hambel et al. is referred to as oxidized IGF-I.
The IGF-I having the primary structure represented by formula (I-b), which has been newly discovered by the present inventors, is referred to as oxidized IGF-I (type B).

This invention relates to a reduced form obtained by recombinant gene method or the like.
When IGF-I is oxidized to produce oxidized IGF-I, the oxidized IGF-I has the same primary structure as IGF-I derived from human blood and is thought to be more naturally accepted when administered to humans.
The present inventors have intensively studied and developed a method for producing IGF-I which would yield more IGF-I (type A) and less oxidized IGF-I (type B).

[Means for solving the problems] The present invention is characterized in that when reduced IGF-I is oxidized in a buffer solution, an organic solvent having solubility in the buffer solution is coexistent with the buffer solution. More specifically, the present invention is characterized in that when reduced IGF-I is dissolved in a buffer solution and left there, and is oxidized by oxygen present in the buffer solution, an organic solvent having solubility in the buffer solution is coexistent with the buffer solution.

In order to carry out the method of the present invention, it is necessary to dissolve reduced IGF-I in a buffer solution.
Conventional techniques commonly used in the field can be used, such as first dissolving -I in a guanidine solution.

The IGF-I solution thus obtained is then diluted with a commonly used buffer solution, such as Tris-HCl buffer, in which case, in the method of the present invention, an organic solvent that is soluble in the buffer solution is also present.

The organic solvent used here is not particularly limited as long as it is soluble in the buffer solution, and preferred examples thereof include lower alcohols with high water solubility such as methanol, ethanol, propanol, and isopropyl alcohol, acetonitrile, dimethyl sulfoxide, and dimethylformamide.

It is also preferable to add a buffer solution when dissolving reduced IGF-I. The pH of the buffer solution for dissolving and diluting reduced IGF-I is preferably 7 or higher, more preferably 7.
The amount of the organic solvent to be present in the reaction system is not particularly limited, and is preferably 20 to 60%, more preferably 30 to 46%.
%.

The oxidation reaction proceeds as an oxidation reaction by oxygen dissolved in the buffer solution when the diluted solution of reduced IGF-I thus obtained is allowed to stand under cooling or room temperature, or, in some cases, under heating at less than about 40° C. Alternatively, a suitable oxidation system commonly used in this field, such as an oxidized-reduced glutathione system, may be used.

The oxidized IGF-I thus obtained has a much higher IGF-I (A type) content than oxidized IGF-I produced in a similar manner using a system without the coexistence of an organic solvent.
Therefore, the method for producing IGF-I of the present invention is consistent with the object of obtaining a larger amount of IGF-I of the same type as IGF-I (type A) isolated from blood.

To isolate oxidized IGF-I (type A) from the reaction product, a method using chromatography commonly used in this field, particularly high performance liquid chromatography (hereinafter referred to as HPLC), is preferred.

EXAMPLES The present invention will be described in more detail below with reference to Production Examples, Comparative Examples and Examples.

Production Example: Production of reduced IGF-I E. coli F-6 (containing plasmid pLHSdM mtrp E. coli HB10
1) (This strain has been deposited at the Fermentation Research Institute, Agency of Industrial Science and Technology under the Budapest Treaty. Deposit number: FERMBP-
729, date of deposit: September 17, 1984) with ampicillin 50 μg/ml
The culture was grown overnight in L broth containing 0.2% glucose, 0.5%
Casamino acids (acid hydrolyzed casein), 50μg/ml vitamin
The solution was diluted 1:20 into M-9 medium containing _B1 and 25 μg/ml ampicillin. β-indoleacrylic acid was added to the medium to a final concentration of 10 μg/ml. The A ₆₀₀ was 0.5.
Next, the mixture was cultured for 2 hours, and the cells were collected by centrifugation (5K rpm, 4° C., 5 minutes).

Wet cell paste (60 g) was dissolved in 150 ml of 10 mM PBS-EDTA (pH 8.0).
The cells were suspended in 0.1 M Tris-HCl (pH 8.0)/8 M urea-0.1 ml and disrupted by sonication. The cell debris was pelleted by centrifugation at 18,000 rpm for 30 min. The pellet was then diluted with 0.1 M Tris-HCl (pH 8.0)/8 M urea-0.1 ml.
Dissolve in M dithiothreitol (50 ml) and rotate at 35,000 rpm for 25
The mixture was centrifuged at 4°C for 30 minutes. The supernatant was removed and diluted with 0.1M Tris-HCl.
The column was loaded onto a Sephacryl S300 Superfine column (trademark: Pharmacia) (5.0 x 86.6 cm, 1700 ml resin) equilibrated with 10 mM 2-mercaptoethanol (pH 8.0)/8 M urea and 10 mM 2-mercaptoethanol. Elution was performed at 4°C with the equilibration buffer at a flow rate of 0.
The flow rate was 6 ml/min. Sephacryl S300 chromatography was performed and 17 ml fractions were collected. Quantitation was performed immediately after fractionation for all chromatographic steps. The active fractions were collected and the combined fractions (255 ml) were dialyzed against 8 liters of 1M aqueous acetic acid at room temperature for 3 hours and then dialyzed overnight against 8 liters of fresh 1M aqueous acetic acid. The dialyzed fractions were lyophilized and the fusion protein containing the desired component was obtained.
The IGF-I was obtained in an amount of 450 mg. The fusion IGF-I was diluted with 15% SDS-PAGE.
GE shows a band at a molecular weight of 15,500.

The fusion IGF-I (225 mg) was dissolved in 36 ml of 60% formic acid. Cyanogen bromide (36 mg) was added and the mixture was reacted under stirring at 25°C or less for 3 hours. After adding 234 ml of distilled water, the formic acid and cyanogen bromide were removed by freeze-drying. The residue was dissolved in 1M Tris-HCl (pH
This solution was dissolved in 36 ml of 0.01 M ammonium acetate (pH 4.6)/8 M urea-50 mM 2-mercaptoethanol.
The mixture was dialyzed twice against 400 ml of M urea-50 mM 2-mercaptoethanol (hereinafter referred to as buffer A) at room temperature for 3 hours, and then dialyzed against 400 ml of fresh buffer A overnight.

The dialysis solution is a cation exchange resin equilibrated with buffer A.
The column was loaded with CM52 (1.6 x 7.5 cm, 15 ml resin) and was loaded with Buffer A (60 ml) at room temperature at a flow rate of 0.25 ml/min.
The solution was washed for 10 min with 0.2 M ammonium acetate/8 M urea-50 mM 2-mercaptoethanol (120 ml) from buffer A.
The fractions were eluted with a linear gradient from No. 56 to No. 61.
2.9 ml/fraction was collected.

The collected fractions were subjected to HPLC under the following conditions.

Column: Ultrapore RPSC (4.6 x 75 mm) (trademark: Beckman) Flow rate: 1 ml/min Elution: Linear gradient of 10% to 60% acetonitrile in 0.1 M trifluoroacetic acid; 50 min Chromatography was repeated 15 times to collect fractions containing reduced IGF-I. The main peak at retention time 29.32 min was reduced IGF-I.
The reduced IGF-I obtained by the above procedure corresponds to
The mixture was freeze-dried to obtain about 2.4 mg of powder.

Comparative Example Reduced IGF-I obtained in the same manner as in the above Production Example was dissolved in 6M guanidine/0.05M Tris-HCl buffer (pH 8.0),
The concentration was 3.3 mg/ml. 1 ml of this solution was diluted with 0.05 M Tris.
After diluting with 11 ml of hydrochloric acid buffer (pH 8.0), the mixture was incubated at room temperature for 7
After removing the insoluble matter with a 0.45 μm filter, the contents of the solution were measured by HPLC under the following conditions. The results were: 0.93 mg of oxidized IGF-I (type A), 0.35 mg of oxidized IGF-I (type B),
It was found that each contained mg.

[HPLC-1] Column: Ultrapore RPSC (4.6 x 75 mm)
(trademark: Beckman) Detection method: Absorption at 214 nm Elution method: Linear gradient elution from 10% to 60% acetonitrile in 0.1M trifluoroacetic acid (50 min) Flow rate: 1.0 ml/min Retention time: oxIGF-I (B type) 17.2 min oxIGF-I (A type) 18.2 min The above reaction solution was dialyzed (molecular weight cutoff 3500) against 1 M aqueous acetic acid solution, and then freeze-dried. The resulting powder was
After dissolving in 1 ml of 0.1 M trifluoroacetic acid, HPLC was performed under the following conditions.
C to obtain 0.53 mg of IGF-I (type A).
(Yield 16.2%) [HPLC-2] Column: AP-343-10 (S-10, 200 Å ODS) (20×250 mm) (YMC) Detection method: Absorption at 230 nm Elution: Gradient elution with acetonitrile using a combination of the following buffer a and buffer b.

Retention time: IGF-IB type 15.0 min. IGF-IA type 17.5 min. Example 1 Reduced IGF-I obtained in the same manner as in the above-mentioned production example was dissolved in 6M guanidine/0.05M Tris-HCl buffer (pH 8.0),
The concentration was 3.3 mg/ml. 7.4 ml of 0.05 M Tris-HCl buffer (pH 8.0) and 3.6 ml of acetonitrile were added to 1 ml of this solution to make the final organic solvent concentration 30%, and the solution was left to stand at room temperature for 72 hours. After removing the insoluble matter with a 0.45 μm filter, the solution was analyzed by HPLC in the same manner as in the comparative example, and the result showed that the oxidized form
IGF-I (type A) 1.60mg, oxidized IGF-I (type B) 0.22mg
were found to contain each of the

The reaction solution was diluted 2-fold with 1M acetic acid, and then dialyzed (molecular weight cutoff 3,500) against 1M acetic acid to remove acetonitrile from the reaction solution, and then lyophilized.
The mixture was dissolved in 1 ml of trifluoroacetic acid solution and purified by HPLC under the same conditions as in the comparative example to obtain oxidized IGF-I (A
The reduced IGF-I obtained in the same manner as in the above-mentioned Production Example was dissolved in 6M guanidine/0.05M Tris-HCl buffer (pH 8.0) and 0.94 mg of reduced IGF-I was obtained (yield 28.4%).
The concentration was 3.3 mg/ml. 5.6 ml of 0.05 M Tris-HCl buffer (pH 8.0) and 5.4 ml of acetonitrile were added to 1 ml of this solution to make the final organic solvent concentration 46%, and the solution was left to stand at room temperature for 72 hours. After removing the insoluble matter with a 0.45 μm filter, the solution was analyzed by HPLC in the same manner as in the comparative example, and the result showed that the oxidized form
IGF-I (type A) 1.94mg, oxidized IGF-I (type B) 0.32mg
were found to contain each of the

The reaction solution was diluted 2-fold with 1M acetic acid, and then dialyzed (molecular weight cutoff 3,500) against 1M acetic acid to remove acetonitrile from the reaction solution, and then lyophilized.
The mixture was dissolved in 1 ml of trifluoroacetic acid solution and purified by HPLC under the same conditions as in the comparative example to obtain oxidized IGF-I (A
The reduced IGF-I obtained in the same manner as in the above-mentioned Production Example was dissolved in 6M guanidine/0.05M Tris-HCl buffer (pH 8.0) and 1.14 mg of reduced IGF-I was obtained (yield 34.3%).
The concentration was 3.3 mg/ml. 5.6 ml of 0.05 M Tris-HCl buffer (pH 8.0) and 5.4 ml of methanol were added to 1 ml of this solution to make the final organic solvent concentration 46%, and the solution was left to stand at room temperature for 72 hours. After removing the insoluble matter with a 0.45 μm filter, the solution was analyzed by HPLC in the same manner as in the comparative example. The results showed that the oxidized form
It was found to contain 1.74 mg of IGF-I (type A) and 0.19 mg of oxidized IGF-I (type B).

Example 4 Reduced IGF-I obtained in the same manner as in the above-mentioned production example was dissolved in 6M guanidine/0.05M Tris-HCl buffer (pH 8.0),
The concentration was 3.3 mg/ml. 1 ml of this solution was mixed with 7.4 ml of 0.05 M Tris-HCl buffer (pH 8.0) and 3.
After adding 6 ml of the organic solvent to make the final organic solvent concentration 30%, the mixture was left at room temperature for 72 hours. After removing the insoluble matter with a 0.45 μm filter, the mixture was analyzed by HPLC in the same manner as in the comparative example.
Oxidized IGF-I (type A) 1.14mg, oxidized IGF-I (type B)
They were found to contain 0.18 mg each.

Example 5 Reduced IGF-I obtained in the same manner as in the above-mentioned Production Example was dissolved in 6M guanidine/0.05M Tris-HCl buffer (pH 8.0),
The concentration was 3.3 mg/ml. 1 ml of this solution was mixed with 5.6 ml of 0.05 M Tris-HCl buffer (pH 8.0) and 5.
After adding 4 ml of the mixture to make the final organic solvent concentration 45%, the mixture was left at room temperature for 72 hours. After removing the insoluble matter with a 0.45 μm filter, the mixture was analyzed by HPLC in the same manner as in the comparative example.
Oxidized IGF-I (Type A) 2.03mg, Oxidized IGF-I (Type B)
They were found to contain 0.23 mg each.

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Claims (1)

[Claims] Claim 1: Amino acid sequence: (Wherein, A is alanine, C is cysteine, D is aspartic acid, E is glutamic acid, F is phenylalanine, G is
is glycine, I is isoleucine, K is lysine, L is leucine, M is methionine, N is asparagine, P is proline, Q is glutamine, R is arginine, S is serine, T
is threonine, V is valine, and Y is tyrosine), a reduced human insulin-like growth factor I having the primary structure (In the formula, A, C, D, E, F, G, I, K, L, M,
wherein N, P, Q, R, S, T, V and Y are as defined above, and the three solid lines connecting C and C represent -S-S- bonds, and the oxidized human insulin-like growth factor I (type A) is isolated and collected, the process comprising dissolving an organic solvent having high water solubility in the buffer solution in a reaction system at 20 to 60%.