PT98859B - ENZYME PROCESS FOR THE TRANSFORMATION OF PREPROINSULINS IN INSULINS - Google Patents

Abstract

An internally streamlined suction nozzle (3) carries on its underside a resiliently hinged lever (12) with a catch (13) which enters a recess in the mouthpiece (1) of the appliance. Two lateral circular eyelets (6) near the inlet (3') engage in openings in the floor section (4) which runs on rubber rollers (11). A cover (5) is snapped on to two parallel ribs (9). The nozzle (3) and mouthpiece (1) are quickly separably by upward pressure (16) on the lever (12). USE/ADVANTAGE - On battery- or mains-powered table suction cleaners. The attachment is easily clipped on to appliance and released by thumb pressure.

Description

description

GSP

Insulin consists of two polypeptide chains, the Á chain containing 20 amino acid radicals and the B chain with 30 amino acid radicals. The chains A and B are linked together through two disulfide bridges, with the cysteine radicals at position A 7 s 337 mutually coupled, as well as A20 and 3319. A third disulfide bridge extends between A6 and All. human and animal form in the pancreas in the form of pre-proinsulins. Human pre-proinsulin consists, for example, of a pre-peptide with 24 amino acid radicals, followed by a proinsulin with 86 amino acid radicals with the following configuration:

prépeptido-B-Arg-Arg-C-lys-Arg-A, in which C represents a chain of amino acids with 31 radicals. During the excretion of Langerhans ions, the pre-peptide is de-divided and proinsulin is formed. The C chain is then proteolytically dissociated and active human insulin is formed.

The methods of genetic technology increasingly allow the expression of pre-proinsulin in microorganisms (EP-A 347 781, EP-A-367 163). The dissociation of the “pre-pro” sequences is usually carried out chemically and / or enzymatically (DE-P-3 440 988), EP-A0 264 250). Known methods of enzymatic transformation are based on dissociation with trypsin and carboxypeptidase B (Kemmler W. et al., J. Biol. Chem., 246 (1971) 6786-6791; EP-A-195 691; EP-B- 89 007). The drawback of these methods is the formation of large quantities of by-products which are difficult to separate from the reactive solution. In particular in the transformation of human pre-proinsulin into human insulin (human insulin, Kl) form-HI). This by-product is distinguished from Hl only by the lack of a terminal amino acid and is very difficult to separate from reactive solutions.

To prevent the formation of these by-products, certain heavy metals, especially nickel, can be added to the dissociation preparation (EP-A 0 264 250). A reactive march of this nature is not desirable from the point of view of industrial techniques, due to the high overload of waste water with heavy metals. There remains, therefore, a need for a pre-proinsulin transformation process that is as specific as possible and that is not harmful to the environment.

Clostripain (clostriopeptidase B;

EC 3.4.22.8) is an enzyme from the filtrate of the Clostri dium histolyticum culture with a molecular weight ranging from

about 30,000 to 80,000, which has both proteolytic and amidase esterase activity (Mitchell, W.

M. Harrington, W. F., J. of Biol. Chem. 243 (18), 4683-4692. 1968). It is distinguished by a high specificity for Arg-C bonds. Thus, in the isolated B chain of insulin the Arg-Gly bond is dissociated by clostripain 500 times faster than the Lys-Ala bond and in glucagon only the Arg-Arg, Arg-Ala and lys-Tyr bonds are dissociated. The hydrolysis rates for these bonds are located in the ratios 1, 1/7 and 1/300 (labousse, Β., Bull. Soc. Chim. Biol ·., 42, 1293. 1960). Surprisingly, it has now been found that clostripain dissociates pre-proinsulin specifically at the C-terminal position before arginine, without an appreciable dissociation of the amino acid chain taking place before the arginine in the Β chain (B22).

The invention therefore relates to a process for the hydrolysis of the pre-proinsulin amino acid chain of formula I, (A-1)

Gly-NH (A-6) Cys-S-S (A-20) (A-21) (A-7)

Cys - Cys

Oys - Z - B. (A-ll) (D (B-2)

R ² -R ¹ -HN-Val-Cys (B-7) (B-30)

Oys (B-19)

in which

R ' ¹ ' represents n amino acids, where n is an integer or 1,

R represents hydrogen, a chemically or enzymatically depleted amino acid, or a peptide with 2 to 50 amino acid radicals,

R represents a hydroxy group, an amino acid or a peptide with 2 to 10 amino acids.

X represents L-arginine or a peptide with 2 to 45 amino acids at whose O-terminal and N-terminal end is an L-arginine radical,

Y represents a genetically encodable amino acid, represents a genetically encodable amino acid,

A1 to A20 or B2 to B29 represents a natural amino acid sequence, or modified by exchanging one or more amino acid radicals, human or animal insulin, which is characterized by pre-proinsulin hydrolyzing in the presence of oolestripaine and eventually transform with carhoxypeptidase B into the corresponding insulin.

The amino acid sequence of peptides and proteins is characterized by the N-terminal end of the amino acid chain. Proteases hydrolyze the peptide bond between peptide and protein amino acids. Clostripain especially hydrolyzes peptides or proteins containing L-arginine, after arginine. As products of the pre-proinsulin hydrolysis reaction, derivatives are formed

- 4 R

R ²

insulin or polypeptides that have at the O-terminal end an arginine radical, or amino acids.

The expression natural amino acids means, for example, Gly, Ala, Ser, Thr, Vai Leu,

Ile, Asp, Asn, Glu, Gin, Cys, Met, Tyr, Phe, Pro, Hyp,

Trp, Arg, Lys, Hyl, Orn, Cit or His.

By the expression genetically encoded genetically we mean for example Gly, Ala, Ser, Thr, Vai, Leu, Ile, Asp, Asn, Glu, Gin, Cys, Met, Arg,

Lys, Hys, Tyr, Phe, Trp, Pro or selenocysteine.

Pre-proinsulins of formula I are preferred in which Phe represents, represents hydrogen, a natural amino acid or a peptide with 2 to 30 natural amino acids, ending at the O-terminal end by L-ar & inine, representing a hydroxy group, a natural amino acid , or a peptide with 2 to 10 natural amino acids, represents L-arginine or a carbon chain of a human or animal proinsulin, represents an amino acid of the Thr, Ala or Ser group.

represents an amino acid of the group Asn, Gin, Asp, Glu, Gly, Ser, Thr, Ala or Met.

A1 to A20 or B2 to B29 represent the amino acid sequence of human or animal insulin.

Especially preferred are pre-proinsulins of formula I in which

R stands for Phe,

R represents hydrogen, or a peptide with 2 to 50 natural amino acids that ends at the G-terminus by L-arginine,

R represents a hydroxy group, a natural amino acid, or a peptide with 2 to 10 natural amino acids,

X represents I-arginine or a human, pig or calf proinsulin carbon chain,

Y represents Thr,

Z stands for Asn

A1 to A20 or B2 to B2g represent the amino acid sequence of human, calf or pig insulin.

Insulins that have already been proposed in German Patent Applications P 3 919 852 and P 4 012 818.0 are especially preferred. For example InsuArg with the following amino acid sequence

RHg-Asp Thr Thr Vai Be Glu Pro Asp Pro Asn Ser Asn Gly Arg Phe Vai Asn Gin His Leu Oys Gly Ser His Leu Vai Glu Ala Leu Tyr Leu Vai Oys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thi 'Arg Gly Ile Vai Tyr Glu Gin Cys Cys Thr Ser Ile Oys Ser Leu Gin Leu Glu Asn Tyr Cys Asn-OOOH

Clostripain (EC 3.4.22.8.) Is an extracellular thiolprotease originating from clostridia. The enzyme is a heterodimer and has no homology

with other known thiolproteases. The enzyme-has an exceptionally high specificity for Arg-XXX bonds, especially Arg-Pro. It can be characterized by a molecular weight between 30,000 and 80,000 and an isoelectric point of pH 4.8 to pH 4.9. As activators are active for example cysteine, mercaptoethanol, dithiothreitol or calcium ions.

Clostripain is inhibited in the presence of, for example, tosyl-L-lysine-chloromethyl ketone, hydrogen peroxide, EDTA, Co ^ ⁺ , 0u ^ ⁺ , Cd ^ ^{+ ions} or citrate.

The preparation of clostripain is carried out with the aid of microorganisms, by fermentation. In this process, clostridia are cultivated until the clostripain reaches adequate concentrations in the nutrient medium. It is appropriate, for example, Clostridium mistolyticum, especially Clostridium histolyticum DSM 627. Mutants and variants of the mentioned microorganisms are also suitable, as long as they synthesize clostripain.

The culture is carried out anaerobically, individually or in mixed cultures, for example submerged in static culture, in the absence of oxygen or in fermenters, possibly with supply of nitrogen, noble gases or other gases, except oxygen. Fermentation is carried out in a temperature range from about 10 to 45 ° C, preferably from about 25 to 40 ° C, especially 30 to 38 ° 0. The fermentation is carried out in a range of pK between 5 and 8.5, preferably between 5.5 and 8. Under these conditions, the culture solution generally presents, after 1 to 3 days, an appreciable accumulation of the enzyme. The synthesis of clostripain begins in the late log phase and reaches its maximum in the phase of steady growth. The enzyme production can be monitored with the aid of activity tests (Mitchell W., Meth. Of Enzym. Vol.

(1977) pages. 165-170).

The nutrient solution used for the production of olostripain contains 0.2 to 6% and preferably 0.5 to 3% of organic nitrogen compounds, as well as inorganic salts. As organic nitrogen compounds are of interest: amino acids. peptones, as well as meat extracts, ground seeds, for example corn, wheat, features, soy or cotton, alcohol production distillation residues, meat meal or yeast extracts. Regarding inorganic salts, the nutrient solution may contain, for example, alkali or alkaline earth metal chlorides, carbonates, sulphates or phosphates, iron, zinc and manganese, but also ammonium and nitrate salts.

The optimal fermentation conditions are quite different for each microorganism, but they are either already in the domain of the experts, or they can nevertheless be easily determined with previous tests. Purification of olostripain can be carried out according to classical procedures, for example by precipitation with ammonium sulphate, ion exchange chromatography or gel permeation chromatography. Enzyme coupling is possible according to conventional methods (Oolowick and Kaplan, Meth, Enzymol., Vol. XLIV).

For the enzymatic reaction, whole cells can be used in free or immobilized form, as well as the isolated enzyme product, which can also be attached to supports.

The dissociation of pre-proinsulin of formula I with olostripain is carried out in an aqueous medium to which water-miscible organic constituents, such as alcohols, ketones, urea, N, N-dimethylformamide, can also be added. In particular, corresponding organic or inorganic buffers, such as phosphate, tris, glycine, HEPES, etc., can be added to the reactive preparation, among others, for better control of the reaction pH. The concentration of pre-proinsulin du-

during dissociation, for example, between 0.01 mg / ml and 10 mg / ml, preferably between 0.1 mg / ml and 10 mg / ml. The pre-proinsulin reaction for clostripain (mg for units (U) and is generally 1: 0.01 to 1: 1000, preferably 1: 0.1 to 1:50.

The reaction temperature is also variable over a wide range. A range of temperatures between 0 ° C and -80 ° C is preferred, especially preferably between + 20 ° C and + 40 ° C.

The pH of the reaction can vary between pH 4 and pH 12, with the pH 6 and pE 9 limits being particularly preferred.

The time required for the transformation of pre-proinsulin into the corresponding intermediate compound, depending on the reaction conditions, can vary within a wide range, for example, it can be between 15 minutes and 48 hours, with a reaction time between 1 and 6 hours.

Before use, the enzyme is properly activated in the presence of a mercapto. As mercaptan in this case, in principle, all compounds containing SE groups are preferably used, preferably DTT, DTE, mercaptoethanol, thiolglycellic acid or cysteine.

The concentration of mercaptan can vary within wide limits, with concentrations ranging from 0.1 mM to 100 mM being preferred. In addition, the activation buffer, 2+ contains Ca ions, preferably CaOlg. Activation takes place at pE values between 4 and 12, preferably between pH 6 and pE 8, with the range between pH 7 and pH 8 being particularly preferred. For the convenient maintenance of this pH value, a buffer substance can be added, for example HEPES tris, glycine, etc. The temperature of ac- 9 -

activation can be between 0 ° C and 60 ° 0, with a range of 0 ° C to 10 ° 0 being preferred, and especially preferably 0 to 5 ° 0. The enzyme thus activated can either be used directly or it can be isolated from the activation buffer by chromatography using (^ Ultragel AcA 202.

The dissociation according to the invention of the pre-proinsulins of formula I leads to insulin derivatives with arginine radicals at the O-terminal end of the insulin and to the corresponding dissociated amino acids and / or peptides. Insulin derivatives can, if desired, be transformed into the corresponding insulins with carboxypeptides 3. This can take place in the same reactive preparation, simultaneously with clostripain, but can also occur in sequence under the reaction conditions mentioned above, the insulin derivative being able to eventually be isolated before treatment with carboxypeptidase B, by methods known per se, such as chromatography or crystallization. Carboxypeptidase B can be used in a dissolved or immobilized form. The ratio of carboxypeptidase B to the insulin derivative (weight to weight) is about 1:10 to 1: 5000, but preferably about 1: 500 to 1: 3500 and especially preferably about 1: 1000 to 1 : 3000.

The ratio of carboxypeptidase B to clostripain (weight to weight) is about 1: 1 to 10: 1 and preferably 2: 1 to 5: 1.

The products of the dissociation reaction with clostripain and / or carboxypeptidase B can be prepared for example by precipitation by lowering the pellet value and / or can be purified with the aid of known column chromatographic methods. The insulin obtained can be converted into standard pharmaceutical compositions and can be used as a medicine for the treatment of diabetes mellitus.

In the examples which follow the process of the present invention is described in detail. Percentage indications refer to weight, unless otherwise stated.

Example 1

Closdridium histolytioum DSM 627 is cultured in a nutrient solution with the following composition:

casein-peptone 3% meat extract 30 yeast extract 0.50 cysteine 0.050

KH ₂ P0 ₄ 0.150 pH 7.2.

The pre-culture is inoculated with 10, The culture is carried out in closed flasks under anaerobic conditions. at 37 ° O for about 2 days. The basic preservation of the microorganisms is carried out in the nutrient solution mentioned above with 500 glycerin at -20 ° C. The fermenter is inoculated at 10 ° C with the preculture. Proceed to inoculation in a fermenter with 10 liters of capacity and 8 liters of nutrient solution. The culture is carried out with nitrogen supply at 33 ° C, for 24 hours, and at a constant pH of 7.0. An enzyme activity of 20,000 ϋ / liter was measured in the culture filtrate (Mitchell W., Meth. Of Enzym., Vol.

47, p. 165-170, 1977).

Final isolation was carried out by centrifuging the cells at approximately 6000 g, filtering under sterile conditions through a 0.22 µm pore diameter filter, adding 600 µl of cold methanol (-20 ° C to the filtrate. Subsequently, the solution was kept for 24 hours at -20 ° C and then centrifuged (8,000 g). The centrifugation cake was dissolved in sterile bidistilled water.

and was centrifuged (12,000 g). On the centrifugation cake an enzymatic activity of 300 U / ml, 200 ϋ / mg protein was measured. The yield is 75% of the activity measured in the fermenter.

Example 2

The cell culture was carried out as described in example 1. The production medium in the fermenter consisted of:

peptone protease (Difco) 5 $ cysteine 0.05 $

KK ₂ P0 ₄ 0.15 $ pH 7.2 and inoculated at 2% by pre-culture. Clostripain activity was 45,000 u / ml in the culture filtrate.

Final isolation was performed by tangential flow filtration (langential-Flow-Filtration) on 0.3 _z um membranes (Filtered, Omega Membrane) for cell separation, and by tangential flow filtration on 10 KD membranes (Filtron, Omega Membrane) for the concentration of dissolved clostripain. The concentration factor is 20. Then the concentrate was subjected to the separation of salts and was chromatographed by DEAE-cellulose. Clostripain activity 1000 U / ml; yield $ 85. Storage until use of the enzyme preparation was carried out at -20 ° C.

Example 3

A Activation of clostripina / l of enzyme preparation (200 U / mL, 286 U / mg from Example 1) 1 .mu.l of _z buffer 12 actively -

tion (250 mM DTT, 125 mM CaCl ₂ )

- content in the preparation:

DTT 5 mlí 2.5 mM CaCl ₂ incubation 2 hours on ice

- for the dissociation reaction, the enzyme is diluted 1:40 with tris buffer / 25 mM HCl pH 7.8.

B. Clostripain dissociation preparation for the release of (B31) Arg-insul.ina

100 / il Insu-Arg (1 mg / ml), ul KOI (1 M) _z ul Tris / HCl (1 M, pH 7.8), ul H ₂ 0 _z ul Clostripain (dilution: 1:40)

- content in the preparation:

Insu-Arg 0, 5 mg / ml Clostripain 2, 5 U / rnl DTT 12, 5 uM Tris / HCl 25 mM KOI 100 mM CaCl ₂ 6 one incubation 1-2 hours , 28 ° C;

easily by HPLC, then the reaction with tosyl-L-lysine-chloromethyl-ketone (TLOK) is stopped.

reaoção interruption by adding 1 ul _z TLOK (15 mM) storage at 4 DEG C. result: Human insulin-Arg. With HPLC it is not possible to measure any formation of human insulin (desB30).

- 15 0.

Carboxypeptidase B dissociation preparation for human insulin release

200 µl of clostripain dissociation preparation 10 µl of carboxypeptidase B (dilution 1: 100) content of carboxypeptidase in the preparation: 2.5 µg / ml incubation 2-4 hours at 28 ° C, the reaction is easily as shown by HPLC for the reaction was diluted to carboxypeptidase B (759 U / ml, 150 U / mg, from pig pancreas) to 1: 1000 with tris buffer / HOl 25 pH 7.3 results: human insulin. There is no formation of insulin (desB30) that is detectable by HPLC.

L. KPLG analytical technique xis dissociations were controlled by using a PP 18 column (0.125 M NH ^ (S0 ^) ₂ , adjusted to pH with HgSO ^, 25-50% acetonitrile gradient) or with an 08 column ( 0.1 $ TPA, 20 $ -50 $ acetonitrile gradient).

Example 4

Clostripaina: 200 U / ml (from example 1)

Activation buffer: 500 mM Tris / HOl, pH 7.8

100 mM LTT 25 mM CaCl ₂

Activation; 100 * ul of clostripanin solution> ul of activation buffer

Folding preparation: 35.7 mg insulin S-sulfonate pre-B-chain pre-human A

315 ul 1 M Mercaptoethanol 105 ul 1 M ascorbic acid 100 ml 20 mM glycine buffer, pH 10.7

Folding is carried out overnight in a refrigerator at 40 ° C. The yield of folding is 0.152 mg / ml. After separating the impurities by pH precipitation at pS 5.0, tris is added in a final concentration of 50 mM and 0 pK is adjusted to 7.8 with HCl. 30 µl of the enzyme solution was added. The dissociation took place at 30 ° C and was carried out by means of HPLC.

Results: The pro-proinsulin mentioned above is dissociated from human insulin-Arg. Insulin formation (desB30) is minimal.

Claims (1)

- There Process for the hydrolysis of preproinsulin amino acid chains of formula I - 15 X Cys - Z - R '(A-1) G ly-NH -—--— I (A-6) Cys-S-S (A-20) (A-21) (A-7) Cys — Cys (A-ll) s I s (B-2) R ^Z -R ¹ -HN-Cys - Cys -Y (B-7) (3-19) (B-5O) (D in which R ^ represents n amino acids, where η is the integer or 1, R represents hydrogen, a chemical or enzymatically dissociable amino acid or a peptide with 2 to 30 amino acid radicals, R represents a hydroxy group, an amino acid or a peptide with 2 to 10 amino acids, Σ represents L-arginine or a peptide with 2 to 45 amino acids, at whose C-terminal and N-terminal end is found an L-arginine radical, Y represents a genetically encodable amino acid, Z represents a genetically encodable amino acid, A1 to ASO or B2 to 329 represent a natural amino acid sequence, or modified by exchange of one or more radicals of amino acid, human or animal insulin, characterized by the hydrolysis of preproinsulin in the presence of clostripain and eventually transform with carhoxypeptidase B into the corresponding insulin. Process according to claim 1, characterized in that the preproinsulin of formula I is used in which R stands for Phe, R represents hydrogen, a natural amino acid or a peptide with 20 to 30 natural amino acids, ending at the C-terminus by L-arginine, R represents a hydroxy group, a natural amino acid or a peptide with 2 to 10 natural amino acids, X represents l-arginine or a carbon chain d and a human or animal proinsulin, Y represents an amino acid of the Thr, Ala or Ser group, Z represents an amino acid of the group Asn, Gin, Asp, Glu, Gly, Ser, Thr, Ala or Met, A1 to A20 and B2 to B29 represent the amino acid sequence of human or animal insulin. 3. A process according to claim 1 or 2, characterized in that the preproinsulin of formula I is used in which R ^ · represents Phe, R represents hydrogen, or a peptide with 2 to 50 natural amino acids that ends at the C-terminus by L-arginine, R represents a hydroxy group, a natural amino acid or a peptide with 2 to 10 natural amino acids, X represents L-arginine or a human, pig or calf proinsulin carbon chain, Y represents Thr, Z stands for Asn Ai to A20 or B2 to B29 represent the amino acid sequence of human, calf or pig insulin. 4. A method according to any one of claims 1 to 5, characterized in that the pH is between 4 and 12, preferably between 6 and 9. 5. A process according to any one of claims 1 to 4, characterized in that the concentration of the preproinsulin of formula I is between 0.01 mg / ml and 100 mg / ml, preferably between 0.1 mg / ml and 10 mg / ml. _ 6th Prooesso according to any of claims 1 to 5 characterized in that the preproinsulinaclostripine ratio (in mg for units) is from 1: 0.1 to 1: 1000, preferably 1: 0, 1 to 1:50. - 7. A process according to any of vindications ^J King 1 to 6 wherein the reaction temperature is between 0 and + 80 ° C, preferably between +20 and + 40 ° C. 8. A process according to any one of claims 1 to 7, characterized in that carboxypeptidase B is simultaneously present or used after dissociation with clostripain. _ ga _ Process according to any of claims 1 to 8, characterized in that clostripain and / or carboxypeptidase B are present in an immobilized form The applicant claims the priority of the German application submitted on September 5, 1990, under B2. p 40 28 113.3.