KR20010103511A - Method for preparation of recombinant Guamerin and pharmaceutical compositions containing the recombinant Guamerin for wound healing - Google Patents

Abstract

The present invention relates to a method for preparing a recombinant germline having a wound healing effect and a pharmaceutical composition containing the same, and more particularly, to a recombinant Pichia pastoris having an AOX1 promoter induced by methanol. Development of a dissolved oxygen (STAT) -value fed-batch method using an automatic feedback methanol supply control system to induce the expression of a recombinant germerin with elastase inhibitory activity with high efficiency and a pharmaceutical composition comprising the same as an active ingredient To a red composition. The recombinant germerin of the present invention has an effect of inhibiting chemotaxis of phagocytic cells as an elastase inhibitor, and the pharmaceutical composition containing the same may be usefully used as a therapeutic agent for wound healing.

Description

Method for preparation of recombinant Guamerin and pharmaceutical compositions containing the recombinant Guamerin for wound healing}

The present invention relates to a method for preparing a recombinant protein having a wound healing effect and a pharmaceutical composition containing the same. More specifically, recombinant Pichia pastoris using an AOX1 promoter, in which the expression of an external gene is induced by methanol, Development of a dissolved oxygen (STAT) -value fed-batch method using the automatic feedback methanol supply control system of Pichia pastoris ) to produce a recombinant germinin with elastase inhibitory activity with high efficiency and a pharmaceutical comprising the same as an active ingredient It relates to a composition.

Elastase, known to be present in the monocytes and neutrophils of human leukocytes, is a serine protease that sensitizes important connective tissues, such as elastic fibers or collagen. They are stored in plasma azurophil granules, which are secreted into tissues when they come into contact with necrotic tissues or when microorganisms are affected by phagocytes. If elastase is released uncontrolled at the site of inflammation, it can lead to nonspecific proteolysis, leading to diseases such as emphysema, cystic fibrosis, bronchitis, adult respiratory distress syndrome, asthma, arthritis, psoriasis and pancreatitis (Janoff A. et al., Ann. Rev. Med. 36, 207, 1985). In most cases, the functional activity of elastase in human leukocytes can be modulated depending on the presence or absence of an inhibitor, which may be used as a treatment for semi-inflammatory diseases.

Germerin is an elastase-specific inhibitor of human white blood cells and pig pancreas. It is purified from Hirudo nipponia , a leech in Korea, and consists of 57 amino acids including 10 cysteines. Analysis was made (Jung, HI et al., J. Biol. Chem. 270, 13879, 1995; Kim et al., 1996). In addition, the treatment of pancreatitis caused by germinin in rat models has also been shown to be of interest (Song M. et al., Pancreas 18, 231, 1999).

As such, antiviral effects can be expected since germerin is a potent elastase inhibitor that inhibits the chemotaxis of phagocytic cells, and it is easy to mass-purify the amount of germinin directly from germinin for new drug development. Therefore, as an alternative means to produce a large amount of recombinant germinin having an elastase inhibitory activity using a microorganism has emerged as an important problem.

The biochemical and biological properties of various protease inhibitors isolated from leeches, in addition to the germins, have already been studied. Several expression systems for producing these inhibitors have also been developed because of their therapeutic potential, with hirudin, eglin C and hirustasin intensively studied. In particular, the thrombin inhibitor hirudin isolated from Hirudo medicinalis , a type of leech, has been studied using many expression systems, including Pichia pastoris . Yield was obtained and was successfully purified to a level of 1.5 g / l (Rosenfeld SA et al., Protein Expr. Purif. 8 (4), 476, 1996). In addition, Egrin C, an elastase / cathepsin G inhibitor isolated from Hirudo Medicinalis, was also expressed in E. coli, and its reaction rate and crystallographic analysis were intensively performed (Baici A. et al., Biochem J. 218 (3), 829, 1984; Hipler K. et al., FEBS Lett. 309 (2), 139, 1992). In addition, Hirustacin, known as an antistatin-type protease inhibitor, including antistatin, hirustacin, velastin, piggerin, tain and germinin, was expressed in low yield in Saccharomyces cerevisiae. The structure was revealed by X-ray crystallography (Mittl PR et al., Structure 5 (2), 253, 1998). Considering the structure of the hirustasin revealed by this, it is speculated that many sulfur (-SH) groups form disulfide bonds even in the geranin. In addition, in order to have inhibitory activity, it is necessary to form a correct quaternary structure. In order to form the structure, it is necessary to undergo secretion after expression (secretion), and for this reason, expression using Pichia pastoris , a methylotrophic yeast using methanol, which undergoes the same secretion process as other higher organisms. The system can be usefully used to produce recombinant germinin.

The availability of several systems for the expression of human elastase inhibitors has been previously reported. Expression systems using insect cells and Aspergillus niger showed a yield of 3 to 8 mg / l, and showed complete inhibitory activity against chymotrypsin and leukocyte elastase. The elafin consisting of 57 amino acids derived from human epithelium also exhibited inhibitory activity against leukocyte elastease, and Saccharomyces cerevisiae also used a galactose-induced promoter about 1 mg / It has been reported to be expressed at levels of L and secreted into the medium (Cooley J. et al., Protein Expr. Purif. 14 (1), 38, 1998; Meckelein B. et al., Biomed. Biochem. Acta. 50, 673, 1991; Heinzel R. et al., Eur. J. Biochem. 160, 61, 1986).

However, there have been no reports of any Elastase-specific inhibitors utilizing the Pichia pastoris expression system. However, Wagner et al. Succeeded in mass production of KPI domain of PN-2 / A beta PP using Pichia Pastoris expression system. In this study, 1.0 g / l secreted protein consisting of 57 amino acids was removed from the medium. Expression and purification were able to confirm biochemical properties (Wagner SL et al., Biochem. Biophys. Res. Commun. 186 (2), 1138, 1992).

In addition, many heterologous proteins to date have been expressed and purified in an effective amount that is industrially available using the Pichia pastoris expression system. Generally in the Pichia pastoris system, 60 to 80 of the expressed protein is secreted in a yield of 0.4-4.4 g / l, and 0.3-1.2 g / l is expressed intracellularly (Cregg JM et al., Bio / Technology 11 , 905, 1993; Hollenberg CP et al., Curr. Opinion Biotechnol. 8, 554, 1997). This wide range of yields depends on the fermentation process, the nature of the expression tool and the type of protein of interest.

In the Pichia Pastoris expression system, in order to eliminate genetic instability during the incubator, a method of introducing an expression vector into the chromosomal DNA of Pichia pastoris was introduced. Recently, Pichia pastoris, which produces recombinant germinin, has been prepared (Korean Patent Application No. 98-27344), which is fused with an α-factor leader sequence to secrete the germline structural gene and into host DNA. To be inserted.

In the present invention, the stability of the germline structural gene inserted into the Pichia pastoris chromosome was determined by measuring the number of inserted cassettes, restriction enzyme analysis and DNA sequencing method. Succeeded. In addition, in the Pichia pastoris expression system, an AOX1 promoter, which can use methanol as an inducer, was used as an expression vector. Under the control of the AOX1 promoter, external genes remained unexpressed in the absence of methanol. Using the principle of efficient expression with the replacement of methanol containing media, we have developed our own automatic feedback methanol supply control system for dissolved oxygen (DO) concentrations.

In addition, the present inventors have confirmed that the pharmaceutical composition comprising a recombinant germline produced with high efficiency by the above method as an active ingredient is effective for wound healing and completed the present invention.

Disclosure of the Invention An object of the present invention is to develop a dissolved oxygen (STAT) -value fed-batch method using an automatic feedback methanol supply control system of recombinant Pichia pastoris using an AOX1 promoter in which expression of an external gene is induced by methanol. It is to provide a method for producing a recombinant germerin with high efficiency.

Another object of the present invention is to provide a pharmaceutical composition comprising a recombinant germinin prepared using the above method as an active ingredient, and has a wound healing effect.

1 is a cleavage map of the expression vector (8.2 kb) used for expression of recombinant germinin using Pichia pastoris, showing that the germline cDNA gene (171bp) was inserted between the AOX1 promoter and the AOX1 terminator,

2 is a comparison of the elastase inhibitory activity of the selection clone # 3-32 and # 12-44,

Figure 3 shows the results of the nutritional selection screening label of randomly selected colonies,

Figure 4 shows the result confirmed by Southern blotting the number of expression cassette inserted into the genome of recombinant Pichia pastoris,

Row 1: Size marker (λ / BstEII) Rows 2 and 3: Host strain GS115

Row 4: control strain into which one expression cassette containing the germline gene is inserted

Row 5: Selection strain # 3-32 Row 6: Size marker (λ / BstEII)

Figure 5 shows a cleavage map predicted when two expression cassettes are inserted into the genome of Pichia pastoris,

rG: recombinant germline S: α-factor leader sequence

TT: Terminator 5'AOX1: AOX1 Promoter

*: Mutation

Figure 6 shows a variety of BglII / His4 ( A ), BglII / Gurminer ( B ), EcoRI / His4 ( C ) and EcoRI / Germerin ( D ) when two expression cassettes were inserted into the genome of Pichia pastoris. Southern blotting results using a combination,

A -Column 1: Size markers (λ / BstEII) Columns 2 and 3: Host strain GS115

Row 4: control strain into which one expression cassette containing the germline gene is inserted

Row 5: Selection strain # 3-32 Row 6: Size marker (λ / BstEII)

B -First row: size markers (λ / BstEII)

Row 2: control strain into which one expression cassette containing the germline gene is inserted

Row 3: Selection strain # 3-32 Row 4: Size marker (λ / BstEII)

C -first row: host strain GS115

Row 2: control strain into which one expression cassette containing the germline gene is inserted

Row 3: Selection strain # 3-32 Row 4: Size marker (λ / BstEII)

D -Row 1: Control strain into which one expression cassette containing the germline gene is inserted

Row 2: Selection strain # 3-32 Row 3: Size marker (λ / BstEII)

FIG. 7 shows the results of Southern blotting showing genetic stability in which DNA fragments containing the germline structural genes were observed identically even after repeated cultures up to 70 generations.

Row 1: Number of Households 7.5 Column 2: Number of Households 18.3

3rd column: Number of households 28.6 4th column: Number of households 40.1

Row 5: Number of Households 47.4 Row 6: Number of Households 58.8

Row 7: Number of Households 70.1 Column 8: Size Markers (λ / BstEII)

8 is a graph showing high density cell culture of Pichia pastoris producing recombinant germinin,

9 is a graph showing the time-dependent change in methanol feed rate after induction,

─: Automatic feedback adjustment-: Manual adjustment

Figure 10 is a graph showing the culture pattern over time of the improved recombinant germerin manufacturing method of the present invention,

●: dry colon weight ▼: activity of recombinant germerin

○: residual glycerol concentration ▽: residual methanol concentration

11 is a graph showing the culture pattern over time of the general recombinant germerin production method,

●: dry colon weight ▼: activity of recombinant germerin

○: residual glycerol concentration ▽: residual methanol concentration

12 is a Western blot photograph comparing the accumulation pattern of intracellular and extracellular presynthetic polymerase after induction,

Rows 1 and 10: Recombinant Grain after Purification

2nd, 3rd, 4th, 5th row: total cell pulverization after 0, 6, 18, 25 hours after induction

Row 6, 7, 8, 9: culture supernatant after induction, 0, 6, 18, 25 hours

FIG. 13A illustrates the DO-stat algorithm of the present invention, which illustrates a control mechanism of dissolved oxygen by stirring.

Figure 13b shows the DO-stat algorithm of the present invention, which shows the mechanism of dissolved oxygen by the methanol feed rate,

14 is a graph showing the results of purification of recombinant germinin using hydrophobic binding chromatography ( A ) and reverse phase chromatography ( B ) as absorbance and elastase inhibitory activity,

15 is a graph showing the results of C18 reversed phase HPLC analysis of the recombinant germinin obtained as a result of each purification process,

A: culture supernatant

B: Fraction showing elastase inhibitory activity after hydrophobic binding chromatography purification

C: Fraction showing elastase activity after reverse phase chromatography purification

16 is a graph of binding stoichiometry and inhibition kinetics showing that the recombinant germinin binds to PPE in the same mole number,

17 shows the pH ( A ) and temperature ( B ) stability of the recombinant germerin,

18 is a graph showing a scalpel spectrum using a mass spectrometer of purified recombinant germerin,

19 is a photograph showing visual observation that when the recombinant germinin was added to the fibrin conjugate and applied to the wound in a state where bacterial infection was blocked, the wound healing effect was faster than that of the control group.

20 is a photograph of microscopic observation that the degeneration of the connective tissue is reduced when the recombinant germinin is added to the fibrin conjugate and applied to the wound in a state where bacterial infection is blocked.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for producing a recombinant germerine with high efficiency by developing a DO (dissolved oxygen) -stat fed-batch culture method using an automatic feedback methanol supply control system of recombinant Pichia pastoris .

In the case of the protein of the present invention which must be expressed in large quantities for the development of a wound healing agent, it is not easy to mass-purify the required amount of the protein from leeches. Do. In addition, the germinin contains 10 cysteines in addition to the total 57 amino acids, which, like the germinin, considers the structure of another group of serine protease inhibitors, hirustasin, which is isolated from leeches. Since the -SH) group is assumed to form disulfide bonds, it is important to form the correct tertiary structure for inhibitory activity. Accordingly, the present inventors recombined an expression system using methylotrophic yeast Pichia pastoris , which undergoes secretion, which is the most important process in the formation of the structure of the expressed protein, as well as other higher organisms. It was introduced for production.

Pichia pastoris expression system provided in the present invention, in order to eliminate the genetic instability of the insertion gene caused during the culture, a method of inserting the expression vector into the genomic DNA of Pichia pastoris , High concentration cell culture and mass culture are possible, and various heterologous proteins have been expressed and purified in an effective amount which is industrially available using the same.

In the present invention, in order to express a large amount of germerin that has not been expressed and purified in an industrially effective amount by using the Pichia pastoris expression system, a recombinant blood in which the germline gene is inserted into the genome together with an α-factor leader sequence is first introduced. The genetic characteristics of Kia Pastoris were analyzed. Two copies of the germline structural gene were inserted into the recombinant Pichia pastoris genomic DNA of the present invention, and the inserted germline structural gene was confirmed to be stable up to 70 generations.

Indeed, genetic stability should be ensured throughout the production process, including several times of seed cultivation, in microbial mass expression. Indeed, it should take an average of five to seven generations for cultivation from the master cell bank (MCB), the first cell provided for seed cultivation, to the final cell of the expression stage, and in order to increase the culture scale for large scale fermentation. Many generations do not exceed 20 generations. Thus, ensuring the stability of structural genes up to 70 generations means that continuous culture is sufficiently possible.

Therefore, it was confirmed that high concentration cell culture for generations using recombinant Pichia pastoris was possible, and by using the present inventors, DO-stat fed-batch culture in which the supply of the medium is automatically controlled by the concentration of dissolved oxygen. culture method.

The AOX1 promoter is used in the Pichia Pastoris expression system (Buckholz et al., 1991, Cregg et al., 1993). In the AOX1 promoter system, methanol is preferentially oxidized by alcohol oxidase and used as a carbon source. Alcohol oxidase is not detected in cells cultured using a carbon source such as glucose, glycerol or ethanol, but occupies 30 of the total soluble protein in cells grown in methanol containing media. Thus, foreign genes inserted under the control of the AOX1 promoter remain unexpressed in the absence of methanol, and are efficiently expressed by being replaced with methanol containing medium. As a result, methanol is used as a carbon source and at the same time serves as an inducer.

In addition, dissolved oxygen plays an important role in methanol oxidation by alcohol oxidase (Couderc, R. et al., Agric. Biol. Chem. 44, 2279, 1980). For this reason, the methanol supply method associated with maintaining an appropriate dissolved oxygen (DO) concentration is a very important factor in the preparation of recombinant germinin using the Pichia pastoris expression system of the present invention.

In Pichia pastoris culture, methanol is passed through the oxidative route if only enough oxygen molecules are present, but oxygen deficiency inhibits further oxidation of methanol and accumulates methanol in the culture. At this time, the accumulated methanol can cause fatal toxicity in the cell culture, and at the same time inhibit the expression of recombinant germerin. Therefore, methanol concentration and dissolved oxygen concentration are very important regulators in fed batch batch incubator after induction of Pichia pastoris expression by methanol. The regulation of the expression process by methanol and dissolved oxygen is due to complex interdependencies and cannot be performed efficiently without this interdependence.

In the existing general batch medium feed culture method for the cultivation of Pichia pastoris, the methanol feed was manually varied depending on the DO level used as an indicator. The level of DO was maintained by manual operation of the oxygen mixing and stirring speed during the introduction of air by stirring, and both variables were independently controlled. However, such manual control of methanol supply is not suitable because overfeeding and underfeeding can be caused.

The DO-stat fed-batch culture method using the two correlated circulation systems provided by the present invention is an improvement of the control means to prevent such methanol overexpression and low supply. We applied an automatic feedback methanol feed control system for more sophisticated methanol feed control. This is a means of protecting the cells from oxygen deficiency and excess methanol supply. The method utilizes a computer system to effectively regulate the supply process.

The concept of balanced DO-stat using two correlated circulation systems is applied to the concept of acetic acid emission control in recombinant protein production systems (Konstantinov K. et al., Biotech. Bioeng. 36, 750, 1990). . They adjusted the DO value by changing the stirring speed within a predetermined limit value, and controlled the nutritional supply rate by monitoring the stirring speed, and this efficient nutritional supply made full use of the carbon source. However, there was a problem in applying the procedure directly to the methanol feed of the Pichia Pastoris system.

The conditions required for stable control of DO-stat are

OTR <OUR _max .

OTR is the oxygen transfer rate and OUR _max is the maximum potential value of the oxygen uptake rate, which depends on the physiological state of cell number and biomass concentration. OUR is also closely related to the nutrient consumption rate, which is high when methanol is used as a carbon source due to the high nutrient consumption rate. That is, in an efficient culture process, OUR approaches OUR _max , the maximum possible value of oxygen yield. If DO is regulated by a change in OTR within a narrow range of stirring speeds (1300-1350 rpm), and the feeding rate changes only when the stirring rate is outside the fixed value, the feeding rate and OUR are adjusted to the threshold values of the set stirring speed. Limited by Eventually, the cells grow linearly depending on the OTR value, and the OUR and nutrient yield are set to specific values according to this OTR value. Since the maximum nutritional requirement of the cells and the accompanying OUR _max are closely related to the cell concentration, the greater the cell concentration, the greater the difference between OUR and OUR _max and the efficiency of the expression process. This principle is effective in preventing the accumulation of acetic acid, but is not suitable for the culture of high concentration cells of microorganisms.

The application of the DO-stat principle for the high concentration cell culture of microorganisms can be realized with other simple structures in which DO is directly regulated by the regulation of OUR and OTR. Thus, the present inventors have introduced the concept of DO-stat, which is controlled by simultaneous adjustment of OTR and OUR. In the present invention, the cell concentration of Pichia pastoris reaches 140 g / l at the beginning of the batch culture after the induction of methanol is supplied. In this case, when the oxygen demand is too large, the OTR value is increased even at the maximum stirring speed when aeration is performed by compressed air alone. OUR cannot be maintained. For this reason, the OTR for the DO-stat batch culture process is controlled by changing the mixing ratio of pure oxygen in the air inlet. We at the same time also adjusted OUR by controlling the methanol feed rate. The proposed regulator It is designed to consist of two interrelated circulation systems, and the DO is adjusted to a narrow setpoint (40-45). The first circulation system regulates DO through the regulation of OTR due to the change in the pure oxygen content of the gas inlet (See FIG. 13A). DO value is lower than DO setting value (DO_low), The set value of the pure oxygen ratio at the air inlet is adjusted high, and the DO value is higher than the DO set value (DO_upWhen it rises to), the set value of the pure oxygen ratio in the air inlet is adjusted low. At the same time, the second circulation system regulates the DO value by adjusting the OUR by changing the methanol feed rate.See Figure 13b). If the DO value falls below the DO setting value (DO_lowThis means that methanol is oversupplied, and the methanol feed rate is adjusted to low values. If the DO value rises above the DO setting value (DO_upThis means that there is a shortage of methanol supply, and the methanol supply rate is adjusted to a high value. This circulatory system is measured and regulated every 30 seconds.

Specifically, in the DO-stat fed-batch culture method for recombinant germline production, the cultivation is performed in four steps: inoculum, batch culture, pre-induction and post-induction fed-culture. . First, the inoculum is incubated with YPD medium containing glycerol, and then inoculated with a fermentor containing a minimum salt medium. The pH in the fermenter is maintained at pH 5.1 by the autocontrolled addition of 15 (v / v) NH ₄ OH and 30 (v / v) H ₃ PO ₄ . The dissolved oxygen level is maintained at a saturation of 30 or more by an automatic stirring controller and adjusting the oxygen partial pressure of the inlet gas. The oxygen partial pressure control of the inlet gas is controlled by a computer system using a two-gas mixer at the inlet of air and pure oxygen, and the temperature of the fermentation vessel is maintained at 30 ° C. Cells are incubated for 15 hours in batch culture until all of the glycerol is consumed (cell dry weight> 25 g / l).

Thereafter, DO-stat fed-batch culture is carried out using a glycerol medium containing glycerol and yeast trace metal solution. At this time, when the DO level rises above 50 saturation, the medium supply pump is operated, and when the DO level falls, the operation of the medium supply pump is stopped. This process is repeated by checking DO levels every 30 seconds using Biocommand (New Brunswick Scientific Co.) software. Finally, the medium containing methanol and yeast trace metal solution is fed to induce the production of germinin, the medium feed rate is increased step by step and low medium to activate the expression of the promoter for 4 hours during the initial induction process. Maintain a feed rate.

In the improved DO-stat fed-batch method of the present invention, the first three steps are performed in the same manner as the general batch culture process. However, after methanol induction, DO is controlled in a narrow range of 40-45 in the batch culture feed step. When the DO value falls below the fixed value, the pure oxygen ratio at the air inlet is adjusted high, and when the DO value rises above the fixed value, the pure oxygen ratio at the air inlet is adjusted low. At the same time, when the DO value falls below the fixed level, the methanol supply rate drops, and when the DO value goes up, the methanol supply rate goes up. This circulation system is regulated every 30 seconds. This automatic control utilizes a computer system connected to the fermentor via an interface, and the collected data such as dissolved oxygen, pH, temperature and stirring speed, and nutrient pump speed are analyzed and controlled by the biocommand software.

Recombinant germinin produced by the improved DO-stat batch culture method of the present invention exhibited a higher expression rate of 40 or more compared to the conventional batch culture results in which medium supply was manually controlled. Secreted by the DO-stat fed-batch culture method of the present invention was confirmed that the method of producing a recombinant germerin is efficient.

In addition, the present invention comprises a recombinant germerin prepared using the above method as an active ingredient, and provides a pharmaceutical composition having a wound healing effect. In order to confirm whether the recombinant germerin produced by the above method can be used in a pharmaceutical composition having a wound healing effect, the present inventors have purified the recombinant germinin and revealed its biochemical properties.

1) elastase inhibitory activity

Since Pichia pastoris, which produces recombinant germinin, was cultivated using a chemically distinct medium, the problem of protein contamination from the complex medium was solved. Therefore, a simple purification method using hydrophobic binding chromatography (HIC) and phenyl reverse phase chromatography (RPC) using phenyl sepharose 6FF was used. Purified recombinant germinin was analyzed using C18 reversed phase HPLC, and also confirmed that the purified through SDS-PAGE uniformly. The final purification yield was about 68, and the purification of elastase inhibitory activity was confirmed by 9.3 times after purification.

Elastase inhibitory activity was determined by using pancreatic elastase and N-succinyl-Ala-Ala-Ala-Ala-p-nitroanilide as enzymes and substrates in pigs. One inhibitory unit (iu) is defined as the amount of inhibitor that inhibits the release of 1 μmole p-nitroanilide from the substrate under given conditions. Inhibition constant (Ki) was determined based on tight-binding kinetics and calculated using the following equation.

α = 1-[(E ₀ + I ₀ + K _i )-{(E ₀ + I ₀ + K _i ) ² -4E ₀ I ₀ } ^1/2 ] / (2E ₀ )

Α is a fractional activity of elastase that does not bind to the inhibitor, E ₀ and I ₀ is the initial concentration of the enzyme and the inhibitor, respectively.

2) pH and temperature stability of recombinant germerin

As a result of investigating the stability of the purified recombinant protein to pH and temperature, it showed activity of more than 90 even if left for 24 hours in the range of pH 1-12, and activity of more than 90 even if left for 6 hours at a temperature of more than 90 ℃.

This stability of pH and temperature is expected to be manifested by the rigidity and dense imparted by the many disulfide bonds of the germerin, which means that the recombinant germinin by the production method provided by the present invention forms and expresses the correct structure.

3) In Vivo Immunization of Recombinant Germline

The use of human-derived proteins as drug preparations is an important issue whether or not these proteins elicit an immune response in vivo. Since the recombinant germinin of the present invention does not induce a humoral immune response by itself, it was confirmed that there is no problem in overcoming the limitation of reagent development due to the induction of immune response.

4) Amino Acid Sequencing of Recombinant Germline

N-terminal sequencing, amino acid composition analysis, mass spectrometry was performed to confirm that the purified recombinant germline has the same amino acid sequence and molecular weight as the native germerin. As a result, it was confirmed that the recombinant germinin of the present invention has the same amino acid sequence and configuration as the native germinin. The molecular weight of the recombinant germerin was 6,110 as a result of mass spectrometry, which was the same as that of the native germerin.

Therefore, the recombinant germerin of the present invention has sufficient potential to be used as an active ingredient in the development of new drugs for wound healing. To confirm this, the present inventors actually have a wound healing effect using the pharmaceutical composition including the recombinant germinin. Animal experiments were performed using mice for measurement.

Germerin is a potent elastase inhibitor that inhibits phagocytosis of phagocytic cells, so anti-inflammatory effects can be expected. On the other hand, when bacterial infections occur, phagocytic cells do not have a primary phagocytosis. Rather, the inflammatory condition caused by bacterial infection may worsen. In the present invention, when the recombinant germinin was added to the fibrin conjugate and applied in the state of blocking bacterial infection, it was confirmed that the wound healing effect was faster than that of the control group.

In addition, for the development of new drugs using the recombinant germerin prepared in the present invention, the presence of acute toxicity and antigenicity as part of the safety experiment was confirmed in the ICR mouse, guinea pig and mouse-rat model.

In the acute toxicity study, no animals died during the study in the dose group, and no clinical symptoms occurred. In addition, no significant weight change was observed in the male and female dose groups compared with the control group. In the gross autopsy findings, there were no deaths in the control and dose groups, and no unique autopsy findings were observed in the surviving individuals. Therefore, it can be estimated that the maximum non-toxic amount (harmless dose) by intravenous administration of germerin is 1,000 mg / kg or more.

No specific anaphylaxis symptoms were observed in the results of the active systemic anaphylaxis test for antigenicity. Passive skin anaphylaxis tests were also negative.

In conclusion, the recombinant germinin of the present invention is unlikely to cause adverse effects due to antigenicity in clinical trials, and is not expected to cause a specific anaphylactic response when administered to humans.

The pharmaceutical composition may be administered parenterally during clinical administration and may be used in the form of a general pharmaceutical preparation.

That is, the pharmaceutical composition of the present invention can be administered in various parenteral formulations during actual clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, etc. that are commonly used Is prepared using. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, and lyophilized preparations. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.

In a preferred embodiment of the present invention, in order to block bacterial infection, treatment with fibrin conjugates containing recombinant germinin was performed.

The effective dose of recombinant germinin for wound healing is preferably 1 ng to 100 mg per cm 2 of wound.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1> Confirmation of genetic stability of Pichia pastoris expression system and high concentration culture using the same

<1-1> Preparation of Expression Strains

In order to produce recombinant germins using the Pichia pastoris expression system, first, the germline structural genes obtained by PCR from the germline cDNA library were fused with an α-factor leader sequence including a KEX2 cleavage site to generate a pGS29A expression vector. After the preparation, the strain was transformed into Pichia pastoris GS115, his4 strain to prepare an expression strain using Pichia pastoris (Korean Patent Application 98-27344). The gene was inserted by one intersection between his 4 region in the chromosome and the HIS4 marker of the vector. 1 shows a pGS29A expression vector.

<1-2> Cloning and MCB Preparation

Pichia host strain GS115 is pesto less complex media in YPD (yeast extract 1, 2, peptone, glucose 2), and the transformant was cultured in MD (1.34YNB, 4X10 ^-5 biotin, glucose 2). The sphero flast method was used to transform the PQ29A, a methanol magnetizing yeast, into the expression vector pGS29A. The transformants were plated on MD, a histidine deficient medium, and incubated at 30 ° C. until colonies appeared. Hundreds of selected clones were inoculated in liquid culture solution MGY (1.34YNB, 4X10 ^-5 biotin, 1 glycerol), shaken at 30 ° C and 250 rpm, and cultured at 600 nm for 2-6 OD. when reached, to give change in liquid MM culture medium (1.34YNB, 4X10 ^-5 biotin, 0.5 methanol) to induce protein expression of the transformants, 30 ℃, and incubated 72 more hours with shaking at 250 rpm. The supernatant was taken after centrifugation of the culture, and the primary aging activity was compared.

Enzymes and substrates for measuring elastase inhibitory activity were porcine pancreatic elastase (PPE, Sigma) and N-succinyl-Ala-Ala-Ala-p-nitroanilide (N-Succinyl-Ala-Ala-Ala). -p-nitroanilide) was used. The reaction was performed under buffer of 0.05M Tris-HCl, pH 8.0. Specifically, the same volume of sample solution was added to the buffer solution containing 0.1 unit of PPE, followed by well mixing. The 0.1 mM substrate solution was then added in three volumes, and reacted at 25 ° C. for 10 minutes, and then at 410 nm. The absorbance of was measured. One inhibitory unit (iu) is defined as the amount of inhibitor that inhibits the release of 1 μmole p-nitroanilide from a substrate under given conditions. The ratio () representing elastase inhibitory activity is defined as the reduced ratio of p-nitroanilide produced when 0.05M Tris-Cl (pH 8.0) buffer was used as a control instead of the sample.

As a result, two clones # 3-32 and # 12-24 having the highest elastase inhibitory activity were selected, and the following experiment was conducted for three days of induction of methanol expression for a more accurate comparison. # 3-32 and # 12-24 monoclonal inoculated into minimal medium and incubated for 15 hours. Cells were recovered by centrifugation and then dispersed in a western medium containing 0.5 methanol instead of glycerol. Every 24 hours, methanol was added so that the final methanol concentration of the culture was maintained at 0.5 and maintained for 3 days. In the same manner as in the comparison of the elastase inhibitory activity of strains # 3-32 and # 12-24. Finally, as shown in FIG. 2 , clone # 3-32 showed higher elastase inhibitory activity compared to # 12-44 by methanol induction for 3 days, thus providing clone # 3-32 as a progenitor for seed culture. Was selected for preparation of the master cell bank (MCB).

For MCB preparation, the recombinant Pichia pastoris # 3-32 was incubated for 16 hours in YPD medium, and then the culture solution was mixed 1: 1 with 50 glycerol and transferred to a tube (cryo-tube, Corning). Store at 70 ° C.

To confirm homogeneity in the culture, the phenotype of his4, a nutritional selection marker, of 360 colonies generated from one tube of MCB was analyzed. Colonies from non-selective medium containing L-histidine were transferred to selection medium without L-histidine and incubated at 37 ° C. for 18 hours, resulting in all colonies producing new colonies ( FIG. 3 ). This means that all colonies in the MCB contain the expression cassette uniformly in the genome.

<1-3> Genetic Characterization of Recombinant Pichia Pastoris

Insertion of the cassette containing the germline structural gene was confirmed by analysis of genomic DNA by Southern blotting after restriction enzyme treatment. DNA extraction from recombinant Pichia pastoris was performed using an easy-DNA kit (Invitrogen Co.), and Southern blotting was performed using Bgl II / Gurminer, EcoR I / His4 and EcoR I / Germerin. And various combinations of restriction enzymes and probes. Southern blotting analysis is based on the homologous recombination principle of the his4 region of chromosomal DNA.

Probes to detect the HIS4 and germline genes were prepared by random primer labeling module (Genome Image ^™ , Amersham international plc). Specifically, HIS4 and germinin genes were amplified by PCR using pGS29A vector as a template. The amplified HIS4 and the germline genes were used as a template to bind random primers, and the fluorescein-11-dUTP was included in the DNA probe formed during the random labeling reaction by the Klenow fragment of DNA polymerase I. It was made. DNA probes containing fluorescein-11-dUTP bind to anti-fluorescein antibodies conjugated with alkaline phosphatase (AP), and AP decomposes chemoluminescence (dioxetane) by decomposing the substrate. chemiluminescent signals, which are marked on the film.

Table 1 shows the size of the cleaved DNA fragments expected to be detected by Southern blotting when the expression cassette was inserted by homologous recombination at the his4 position. As a result of Southern blotting, as shown in Table 1 , it was confirmed that multiple genes were inserted. Bgl II restriction enzyme and His4 probe were used to confirm the number of cassettes inserted. As a result, HIS4 structural gene segments were detected at a ratio of 1: 1: 1 in 3.7, 4.7 and 5.8 Kb sized fragments ( FIG. 4 ). The results indicate that two copies of the expression cassette were inserted at his4 position in the Pichia pastoris genome. 5 shows the predicted cleavage map when two expression cassettes are inserted. In addition to the above combination, it was confirmed that various combinations such as BglII / Gurmin, EcoRI / His4, and EcoRI / Gurmin have the same cleavage map as described in FIG . 6 ( FIG. 6 ).

Table 1 Expected DNA Size of Southern Blotting with Various Restriction Enzymes and Probes of Selected Strain # 3-32

Restriction enzyme Bgl II EcoR I probe HIS4 Grain HIS4 Grim Insert 1 copy (kb) 3.7, 4.7 4.7 3.8, 7.4 7.4 Insert 2 copies (kb) 3.7, 4.7, 5.8 4.7, 5.8 3.8, 7.4, 8.2 7.4, 8.2

<1-4> Genetic Stability Analysis of Recombinant Pichia Pastoris

Continuous flask culture was performed to analyze the genetic stability of the germline structural gene inserted into the genome of the recombinant Pichia pastoris prepared above.

Colonies of Pichia pastoris producing germinin were inoculated in 50 ml of YPD medium and incubated overnight with stirring at 30 ° C. and 150 rpm. The culture was used as the inoculum for the next new flask incubation, and continuous culture was repeated up to 70 generations. Each genomic DNA was analyzed by taking 10 ml of culture from each flask just before inoculation. Total DNA of cells was treated with Sal I, electrophoresed on 0.6 agarose gel and then transferred to nylon membrane for blotting. Probe for the structural gene of the germerin was prepared using a random primer labeling module (Genome Images ^TM , Amersham International plc).

As a result, as shown in Fig . 7 , DNA fragments containing the structural genes of the germline were observed in the same manner even after repeated cultures up to 70 generations. Inserted structural genes for the production of highly efficient synthetic proteins should be stable throughout the production process, including several seed cultures. In general, the preparation of working cell banks (WCBs) expressing the actual recombinant protein from the MCB, which is a producer, usually takes five to seven generations, and for large-scale fermentation, even if the culture scale is increased, more than 20 generations. It does not exceed Thus, as confirmed above, if the stability of the structural genes up to 70 generations is ensured, it can be said to be sufficiently industrially available. After all, the continuous culture results indicate that the stability of the germline structural genes required for industrial production is ensured.

<1-5> High concentration culture of recombinant Pichia pastoris

For high concentration cell culture for the production of germline of recombinant Pichia pastoris whose genetic stability was confirmed, the present invention used a DO-stat batch medium supply method by inducing methanol. Using a Bioflio III fermenter (New Brunswick Scientific Co.), temperature, pH and air saturation were incubated at 30 ° C., 5.0 and 2.0 vvm, respectively. First, in order to prepare the inoculum, seed culture was performed for 18 hours at 30 ° C. and 200 rpm in 50 ml YPD medium. Minimal salt medium was used for the culture containing yeast trace metal solution. After glycerol was completely depleted in the medium, fed-batch culture was performed continuously. The feed of medium above was controlled at 50 air saturation conditions. The feed medium contains 50 (w / v) glycerol and 12 ml / l yeast trace metal solution. After 500 ml of medium was fed, the feed of the medium was stopped and further incubated for 0.5 hours to completely deplete glycerol from the culture. Finally, a medium containing 50 methanol and 12 ml / l yeast trace metal solution was fed to induce the production of germinin, with the medium feed rate being stepwise increased for 40 hours from 22 to 46 ml / h.

As a result, as shown in Figure 8 , the cell concentration after the DO-stat fed-batch culture appeared to have an absorbance of 250 at 600 nm. The change in cell concentration was also observed in methanol supply. Recombinant germinin was secreted into the medium up to 260 mg / l after methanol induction, and finally the number of generations of high concentration cell incubator producing recombinant germinin was 10.4 generations. During the induction period, the number of generations increased by 0.1. This result means that the expression cassette is stably maintained not only in the growth phase but also in the induction phase.

In order to confirm that the DNA sequence of the germline structural gene was correctly maintained even after high concentration of cell culture, three DNA sequencing analysis was repeated.

Samples for DNA sequencing include MCB, final culture and EOPC (high concentration cell culture).

Liquid) was used. Primers corresponding to 5'AOX and 3'AOX nucleotide sequences were used for PCR amplification of the germline gene, and the amplified germline gene was transformed using a pGEMT easy transformation kit (Promega). As a result of nucleotide sequence analysis of the DNA extracted from the transformant, it was confirmed that the DNA nucleotide sequences of all the samples were identical to the nucleotide sequences of the expression vectors. These results reaffirm the stability of the inserted germline gene.

Example 2 Production of Recombinant Germinin Using an Improved Pichia Pastoris Expression System

In the DO-stat fed-batch cultivation of the improved Pastoris expression system used in the present invention for the production of recombinant germline, the cultivation is carried out in four stages: inoculum, batch, pre-induction fed-batch (pre- induction fed batch and post-induction fed batch. Incubation of the inoculum was performed by adding 100 μl of glycerol to a flask containing 100 ml of YPD medium (1 (w / v) glucose, 1w / v yeast extract, 2 (w / v) peptone), followed by 30 ° C. and 150 rpm. Incubated for 20 hours at. The cultured inoculum contains 2.5 L of minimal salt medium (glycerol, 40 g / L; H ₃ PO ₄ , 27 mL / L; CaSO ₄ , 0.9 g / L; K ₂ SO ₄ , 18.0 g / L; MgSO ₄ Bioflio III fermenter (New Brunswick Scientific Co.), containing 10.24 g / l; KOH, 4.13 g / l; MH ₄ OH, 30 ml / l; yeast trace metal solution, 4.4 ml / l) .) Was inoculated. Yeast trace metal solution is CuSo ₄ · 5H ₂ 0 6.0 g / l, KI 0.09 g / l, MnSO ₄ · H ₂ O 3.0 g / l, NaMo.H ₂ O 24.0 g / l. H ₃ BO ₄ 0.02 g / l, CoCl ₂ 0.5 g / l, ZnCl ₂ 20.0 g / l, FeSO ₄ H ₂ 0 65.0 g / l, biotin 0.2 g / l, H ₂ SO ₄ 20 ml / l do. The pH in the vessel was maintained at pH 5.1 by the autocontrolled addition of 15 (v / v) NH ₄ OH and 30 (v / v) H ₃ PO ₄ . Dissolved oxygen levels were maintained above 30 saturation by automatic stirring controller and oxygen partial pressure of inlet gas. The oxygen partial pressure control of the inlet gas was controlled by a computer system using a two-gas mixer (New Brunswick Scienctific, Edison) at the inlet of air and pure oxygen. The temperature of the fermentation vessel was maintained at 30 ℃. Cells were incubated for 15 hours in batch culture mode until all glycerol was consumed (cell dry weight> 25 g / l). Thereafter, DO-stat fed-batch culture was performed using a glycerol medium containing 80 (w / v) glycerol and 12 ml / l yeast trace metal solution. When the DO level rises above 50 saturation, the feed pump is turned on. When the DO level drops, the feed pump stops working. This process was repeated by checking DO levels every 30 seconds using Biocommand (New Brunswick Scientific Co.) software. During the glycerol DO-stat fed-batch cultivation, 500 ml of medium was fed.

Finally, a medium containing 100 methanol and 12 ml / l of yeast trace metal solution was fed to induce the production of germinin, with the medium feeding rate being stepwise increased for 40 hours from 22 to 46 ml / h. During the initial induction process, a low medium feed rate was maintained for 4 hours to activate the expression of the promoter.

In the improved DO-stat fed-batch of the present invention, the first three steps were performed in the same manner as the general batch culture process. However, in the post-incubation step DO is controlled in a narrow range of 40-45. When the DO value falls below the fixed value, the pure oxygen ratio at the air inlet is adjusted high, and when the DO value rises above the fixed value, the pure oxygen ratio at the air inlet is adjusted low. At the same time, when the DO level falls below the fixed level, the methanol supply rate drops. When the DO level goes up, the methanol supply rate goes up. This circulation system is regulated every 30 seconds.

The Pentium 133 MHz PC system was used for the measurement, calculation and adjustment of the data of the present invention. The computer was connected to the fermentor via the RS422 interface. Collected data such as dissolved oxygen, pH, temperature and agitation rate, and nutrient pump rate were analyzed by BioComment software.

In analyzing the results of the culture method, the cell concentration was determined by measurement of the cell dry weight. To this end, 10 ml of the culture was centrifuged at 9000 rpm, and the precipitate was washed twice with phosphate buffer solution. Finally, the cell precipitate was dissolved in distilled water and dried using an electronic moisture analyzer (Sartorius MA 40, Sartorious).

Glycerol and methanol concentrations were measured by gas chromatography (HP5890 series II, Hewlett Packard), using 6 ft OV-17 WHP 100/120 19001A-B12 and 6 ft10degs whp 100/120 19001a-L12 columns.

Western blot analysis was performed on anti-recombinant germline serum from rabbits for determination of secretion efficiency. Cells and culture supernatants collected at regular time intervals were analyzed. In the present invention, the electrophoresis was moved to the membrane, treated with glutaraldehyde to bind the peptide, thereby enhancing the detection sensitivity (Karey and Sibasku, Anal. Biochem. 178, 255, 1989), wasabi kinase Analysis was performed using conjugated secondary anti-goat rabbit IgG.

The results of the production method of the present invention devised as described above were compared and analyzed with the results of the oil price cultivation method in which the medium supply was manually controlled.

As shown in Figure 9 , the proposed two regulating circuits were adjusted simultaneously and applied well to the computer system, so after initial setup, there was no need to change the parameters. An improved culture of DO-stat is shown in FIG. 10 . Recombinant germline expression level after 24 hours after the culture was 450 U / ℓ, which is 40 high value compared to the result of the conventional conventional fed-batch culture method shown in FIG . At this time, Western blotting results show that there is almost no recombinant germinin in the cells of the production phase ( FIG. 12 ). This means that recombinant germinin is secreted into the medium immediately after expression, and in the expression system of the present invention, the secretion mechanism of recombinant Pichia pastoris is well established.

In conclusion, as a result of using the DO-stat fed-batch culture method for the production of the recombinant germline of the present invention, the production system of the recombinant Pichia pastoris was well established and the yield of the recombinant germline was increased. I could confirm it.

Example 3 Separation and Purification of Recombinant Germerin

<3-1> Isolation of Recombinant Germinin Using Chromatography

In order to confirm whether the recombinant germinin produced using the DO-stat fed-batch culture method of the present invention can be actually used for drug development, the recombinant germinin was isolated. For hydrophobic interaction chromatography, ammonium sulfate was first added to a final concentration of 1.0 M to the culture supernatant containing the expressed recombinant germinin. The solution was filled with Phenyl Sepharose 6FF (Amersham Pharmacia Biotech.) And injected into an HIC column (Econo, 5 cm x 13 cm, Biorad) equilibrated with 0.05 M Tris buffer containing 1.0 M ammonium sulfate, 1.0 Protein was eluted by injecting 1.5 L of a buffer solution containing ammonium sulfate at a concentration gradient of -0.0 M at a rate of 50 mL / hr. The eluate containing the recombinant germerine was filtered using a 0.2 μm filter and reversed phase chromatography using C8-100 10 SP (2.5 cm × 10 cm, Amicon) equilibrated with 2 (v / v) 2-propanol. Was performed. Elution was performed using a 2-propanol / water concentration gradient (2-5-20-95 (v / v) at a rate of 30 mL / hr. The purified eluate was 280 nm using a UV detector (Beckman). The absorbance at was analyzed and the elastase inhibitory activity was determined.

As a result, elastase inhibitory activity was measured in fractions eluted with 0.2 M ammonium sulfate ( FIG. 14A ). The impurities in the culture were almost completely removed using HIC as shown in FIG. 15 . Elution fractions from HIC were again purified using RPC. Fractions showing elastase inhibitory activity were fractions obtained at an initial concentration gradient of 2-propanol / water ( FIG. 14B ). Purified recombinant germerine was confirmed to be uniformly purified through C18 reverse phase HPLC and SDS-PAGE. Reverse phase HPLC was performed at a column temperature of 40 ° C. with a solvent of acetonitrile / water containing C18 column (Macrosphere 300 C18 5U, 250 mm × 4.6 mm, Altech) and 0.1TFA (tetraflouroacetic acid) at a rate of 0.7 μl / min. The concentration was maintained at 10 acetonitrile for 5 minutes, and then the concentration gradient was changed to 35 for 5 minutes, 55 for 10 minutes, and 95 for the last 5 minutes. The fraction was then dried at low temperature for analysis, and the final purification yield was about 68.

Elastase inhibitory activity was measured using the method described in Example 1 to determine the specific activity of the recombinant germerin. Inhibition units calculated from the specific inhibitory activity of the recombinant germerin are shown in Table 2 .

TABLE 2 Post-purification activities of recombinant germerin

The elastase inhibitory activity of the recombinant germerin of the present invention was 603 u / g, and it was confirmed that the activity after purification was increased by 9.3 times or more. This indicates that the amount of recombinant germline in the culture accounts for 11 of the total protein. Inhibition kinetics of recombinant germerine is a reversible co-molecular binding inhibitor of PPE with a Ki of 38 nM. In addition, Fig. 16 shows binding stoichiometry and inhibition kinetics, indicating that recombinant germinin binds to PPE in the same mole number.

Thus, two steps of chromatographic processes (HIC and RPC) resulted in the production of 98 purity recombinant germinin with a total inhibitory activity in 68 yield.

<3-2> pH and temperature stability of recombinant germerin

In order to investigate the stability of the purified recombinant protein to pH, after remaining at 25 ℃ for 0 to 24 hours in the range of pH 1 to 12, the residual elastase inhibitory activity was determined. In order to investigate the temperature stability, it was left for 0.5 to 6 hours at 50, 70, and 90 ℃, and then the elixase residual activity was measured. As a result, in the pH and temperature stability, even if left for 24 hours in the range of pH 1 to 12 shows an activity of 90 or more ( A of Fig. 17 ), even if left for 6 hours at a temperature of 90 ℃ or more shows more than 90 activities. It was confirmed ( B of Fig. 17 ). These results indicate that the stiffness and dense imparted by the many disulfide bonds of the germinin indicate stability of pH and temperature.

<3-3> Amino Acid Sequence Analysis of Recombinant Germline

N-terminal sequencing, amino acid composition analysis, and mass spectrometry were performed to confirm that the purified recombinant germline has the same amino acid sequence and molecular weight as the native germerin. N-terminal sequencing was performed using an automated peptide sequencer (Model 492, Procise ™, Perkin-Elmer). For amino acid composition analysis, recombinant Germerin was hydrolyzed at 155 ° C. for 60 minutes in 6 N HCl and washed. After drying, the mixture was derivatized with PTC (phenylisothiocyanate). The PTC-amino acid was isolated using a C18 reversed-phase column (Bidlingmeyer BA et al., J. Chromatogr. 336 (1), 93, 1984), and the molecular weight of the recombinant germerin was determined by using a mass spectrometer, Platform II. II, Micromass) (Van Dorsselar A. et al., Biomed. Environ. Mass Spectrom. 19 (11), 692, 1990).

As a result, it was confirmed that the recombinant germinin of the present invention has the same amino acid sequence and configuration as the native germinin ( Table 3 ). The molecular weight of the recombinant germerin was 6,110 as a result of mass spectrometry, which was the same as that of the native germerin ( FIG. 18 ).

Table 3 Analysis of Amino Acid Composition of Purified Recombinant Germline

*: Cysteine residues cannot be quantitatively analyzed due to the limitation of the analysis method itself.

<3-4> Recombinant Germinin Immunization

In order to investigate the immunization reaction of the recombinant germerin, four samples were administered by intraperitoneal injection to investigate antibody production in mice. As a negative control group, a phosphate buffer solution and an adjuvant were used, and as an experimental group, 500 μg of each phosphate buffer solution and an adjuvant, including recombinant germerin, were used. Each sample dose was administered to 5 mice.

Mice were harvested blood one, two or four weeks after the first immunization and the second immunization was performed at the end of two weeks after the first immunization. Antibody titers of each group were quantified by enzyme-linked immunoassay (ELISA) (Tijssen, P., Elsevier Science Publishers BV, The Netherlands, 1985), and each titer was mouse negative control serum absorbance at mean absorbance. Defined as the degree of dilution of the positive sample determined by the cut-off value plus three times the standard deviation.

As a result, the recombinant germinin itself did not elicit a humoral immune response except when administered with an adjuvant ( Table 4) .

Table 4 In Vivo Immune Responses of Recombinant Germline in Mice

Therefore, the prepared germerin of the present invention does not induce a humoral immune response, it was confirmed that there is no problem in overcoming the limitations of the reagent development due to the immune response.

Example 4 Wound Healing Effect of Recombinant Germerin

Animal experiments were performed using mice to determine the effect of wound healing using the pharmaceutical composition comprising the recombinant germerin of the present invention. Germerin is a potent elastase inhibitor that has the effect of inhibiting the chemotaxis of phagocytes, so anti-inflammatory effects can be expected. Based on this fact, the present invention observed the wound healing process in the absence of bacterial infection.

In order to examine the wound healing process in the germ-free state, a small area of movement in the back of the mouse, usually the back of the chest, the skin was treated with povidone and alcohol disinfectant, and then cut in about 7 mm with a surgical knife 15, and then fibrin junction It was applied to the agent (green cross) and treated to prevent bacterial infection after dissection. In the control group, a pure fibrin conjugate was used, and in the experimental group, the recombinant germerin purified in Example 3 was diluted and used at a concentration of 100 ng / ml. Twenty mice were used in all, and four mice were examined visually and histologically after 1, 2, 3, 4, and 5 days after incision.

As a result, as shown in Figure 19 , when used in addition to the fibrin conjugates, it showed a faster wound healing effect than the control group. In particular, in the naked eye, the control group showed a large open wound and delayed healing. However, when the Kirinin was applied to the wound, the wound did not open, and the healing pattern was gradually observed. In microscopic observation, dissolution of collagen fibers was observed in the control group due to moderate inflammatory response and degeneration of the connective tissue. It could be confirmed ( Fig. 20 ).

Example 5 Safety Experiments of Recombinant Germline

As part of the safety experiment to develop a new drug using the recombinant germerin prepared in the present invention, to confirm the presence of acute toxicity and antigenicity in the ICR mouse, guinea pig and mouse-rat models, the Korea Food and Drug Administration notice (1998) 12. 3. Active systemic anaphylaxis test and passive skin anaphylaxis reaction test during acute toxicity and antigenicity test in accordance with No. 1998-116. cutaneous anaphylaxis test, PCA test).

<5-1> Acute Toxicity Test

Recombinant germinin was used by suspending the form of lyophilized white powder in sterile physiological saline (foreign pharmaceuticals) with 2-propanol / water as the final solvent. In the antigenicity test, albumin (ovalvumin, Sigma) was dissolved in sterile physiological saline as a positive control and mixed with an adjuvant (Freund's complete adjuvant, Gibco) in the same amount. Used.

17-24 g SPF (specific pathogen free) ICR mice in the acute toxicity test, 200-350 g Hartley male guinea pig, 20-30 g Balb / c male mouse, 250-270 g Male Sprgue Dawley rats were purchased at Seoul National University's experimental animal breeding center and allowed to acclimate for about one week, and then healthy animals were selected. Temperature 23 ± 3 ℃, relative humidity 50 ± 10, exhaust 10-12 times / hour, fluorescent light intensity 12 hours. Cycle, roughness 150-160 Lux was tested in a polycarbonate rearing box for the entire test period. During the purifying and testing period, solid feed for experimental animals was purchased from Sinchon Feed Co., Ltd., and drinking water was allowed to freely drink tap water.

First, weights were measured for animals determined to be healthy during the acclimation period, and groups of individuals close to the average body weight were selected and randomized. It was confirmed by performing a pharmacological verification. Individual identification of animals was performed using the pigmented pigmentation method and the breeding and tagging tag labeling method. Each group consisted of 10 males and 5 males and 10 females.The dose was set to 1,000 mg / dose in consideration of preliminary results and solubility of the test substance. Administered

Recombinant polymer was diluted with distilled water on the day of the test to a concentration suitable for the test group, and then injected into the vein of the mouse using a 1 ml syringe. The dose setting was calculated by measuring the weight immediately before intravenous administration.

Clinical symptoms of all test animals were observed at least once a day for 1 hour to 6 hours after administration on the day of administration and at least once daily for 1 to 14 days of administration. Careful observation was made of the presence of a dead animal and the symptoms likely to be caused by the test substance after administration of the test substance.

All experimental animals used in the test were weighed on the day (0 day), 4th, 7th, 12th and 14th day of administration of the test substance.

Survival cases after the end of the test were weighed before necropsy and bleeding under ether anesthesia, and the appearance and internal organ abnormalities were observed in detail.

Statistical analysis was performed using ANOVA (one way analysis of variance), and the significance level between the control group and the control group was tested at the levels of P <0.05 and P <0.01.

The acute toxicity test showed no animals died during the study in the dose group ( Table 5 ) and no clinical symptoms ( Table 6 ). In addition, no significant weight change was observed in the male and female dose groups compared to the control group ( Table 7 ). In the gross autopsy findings, there were no deaths in the control and dose groups, and no unique autopsy findings were observed in the surviving individuals ( Table 8 ).

<Table 5> Mortality after Intravenous Injection

gender Dose (mg / kg) Mice Time after dosing Days after dosing Final mortality 1 2 3 4 5 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 cock Control 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5 1,000 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5 female Control 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5 1,000 5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0/5

<Table 6> Clinical Symptoms after Intravenous Injection

gender Dose (mg / kg) Clinical symptoms Days after dosing Start 1 2 3 4 5 6 7 8 9 10 11 12 13 14 cock Control NAD 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1,000 NAD 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 female Control NAD 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1,000 NAD 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

NAD: No abnormal clinical symptoms observed.

Table 7 Body weight after intravenous injection

gender Dose (mg / kg) Number of mice Days after dosing 0 4 7 12 14 cock Control 5 Average S.D. 23.403.21 24.402.51 27.201.30 28.201.10 28.200.84 1,000 5 Average S.D. 23.600.89 26.002.12 27.801.92 28.001.58 28.002.24 female Control 5 Average S.D. 17.802.59 23.401.82 25.602.30 25.201.48 25.400.89 1,000 5 Average S.D. 17.800.84 25.000.71 25.801.48 25.600.89 25.801.10

p <0.05 showed significant difference from control.

p <0.01 showed significant difference from control.

S.D. : Standard Deviation

Therefore, it can be estimated that the maximum non-toxic amount (harmless dose) by intravenous administration of germerin is 1,000 mg / kg or more.

<Table 8> Autopsy Findings after Injecting Germerin

Autopsy findings cock female Dose (mg / kg) Control 1,000 Control 1,000 Mice 5 5 5 5 Adrenal Cortex NGF Brain NGF Heart NGF Liver NGF Kidney NGF Interest NGF Testicular NGF Ovary NGF Thymus NGF 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100) 5 (100)

NGF: No specific autopsy findings observed.

(): Ratio of unobserved specific autopsy findings

<5-2> Active Systemic Anaphylaxis Response Test

The dose setting and dosing schedule of the germerin used for sensitization are shown in Table 9 .

Table 9 Sensitization of Guinea Pigs for Active Systemic Anaphylaxis Response Tests

group Substance Dosage time Number of individuals Dosing method I Ⅱ Ⅲ Ⅳ Ⅴ Grim Grim Grain + FCA^cOVA^d+ FCA voice control 10 μg / object 100 μg / object 100 μg / object 2 mg / object 1 ml / object 9 ^a 9 ^a 3 ^b 3 ^b 9 ^a 7 7 7 7 7 Subcutaneous administration Subcutaneous administration Subcutaneous administration Subcutaneous administration Subcutaneous administration

a: 3 times a week

b: 1 time per week for 3 weeks

c: Freund's complete adjuvant

d: albumin

In 7 guinea pigs per group, group 1 was 10 µg / object, group 2 was 100 µg / object, group 3 was 100 µg / object + FCA, group 4 was albumin (ovalvumin) 2 mg / object + FCA, 5 The group was administered as a negative control (vehicle). Subcutaneous administration was performed three times a week for the single group and three times for the mixed group once a week. The test materials were all dissolved in sterile saline, and two weeks after the last sensitization, the induced antigen (100 μg / object or albumin 10 mg / object) was administered into the tarsal vein. Systemic symptoms were observed for 30 minutes after the induction to confirm the presence of anaphylactic shock, and the test results were evaluated according to the criteria of Table 10 .

Table 10 Criteria for active systemic anaphylactic reaction test

Anxiety 2. Hair growth 3. Tremor 4. Rub or lick nose 5. Sneezing 6. Cough 7. Nervousness 8. Urination 9. Bowel movement 10. Yun, tears 11. Shortness of breath 12. Bully 13. Cyanosis 14. Walking anxiety 15. Skipped 16. Gasping 17. Convulsions 18. Lateral and (19) 19. Cheyne-Strokes Breathing 20. Dead Judgment [-] Asymptomatic [±] Weak Symptoms: 1 to 4 [+] Moderate Symptoms: 1 to 10 [++] Severe Symptoms: 1 to 19 [+++]

The results of the active systemic anapalax reaction test are shown in Table 11 . In the group sensitized with 10 μg / individual and 100 μg / individual alone and 100 ㎍ / individual mixed with the adjuvant, only two of the mixed sensitized groups showed false positive, and the rest of the groups showed unusual ana There were no symptoms of palaxis. Severe anaphylactic reactions were observed in all animals when sensitized with albumin as a positive control.

Table 11 Results of Active Systemic Anaphylaxis Response Test

group Sensitization Reaction time Anaphylaxis Reaction-± + ++ +++ Positive reaction rate I (10 μg / object) Germerin (100 μg / object) 7 ----- 0/7 Ⅱ Germerin (100 μg / object) Germerin (100 μg / object) 7 ----- 0/7 Ⅲ Grain + FCA ^a (100 μg / individual) Germerin (100 μg / object) 7 - 2 - - - 2/7 Ⅳ OVA ^b + FCA (0.2 mg / individual) OVA (10 mg / individual) 7 ----7 7/7 Ⅴ Negative control group (0.1 ml / object) Germerin (100 μg / object) 7 ----- 0/7

a: immunoadjuvant

b: albumin

<5-3> Passive skin anaphylaxis reaction test

The dose and group setting of the germinin used for sensitization was similar to that of the active systemic anaphylaxis shock response test. In the seven male Balb / c mice per group, group 1 was 10 µg / object, group 2 100 μg / object, group 3 was administered 100 μg / object + FCA, group 4 was albumin (ovalvumin) 0.2 mg / object + FCA, and group 5 was administered as a negative control (vehicle) ( Table 12 ).

Table 12 Sensitization of Mice for Passive Skin Anaphylaxis Response Test

group Substance Dosage time Number of individuals Dosing method I Ⅱ Ⅲ Ⅳ Ⅴ Grim Grim Grain + FCA^cOVA^d+ FCA voice control 10 μg / object 100 μg / object 100 μg / object 0.2 mg / object 0.2 ml / object 9 ^a 9 ^a 3 ^b 3 ^b 9 ^a 7 7 7 7 7 Subcutaneous administration Subcutaneous administration Subcutaneous administration Subcutaneous administration Subcutaneous administration

a: 3 times a week

b: 1 time per week for 3 weeks

c: Freund's complete adjuvant

d: albumin

Subcutaneous administration was performed three times a week for the single group and three times for the mixed group once a week. The experimental group, the amount of sensitization, the number of sensitization, and the sensitization path are shown in Table 3, and two weeks after the final administration, blood was collected from the mouse orbital vein and serum was isolated. Remove the rat's back hair as wide as possible using the clipper, mark the left and right areas of the back with red magic in advance, and then 100 ml of mouse antiserum diluted 8 to 6 steps with sterile saline solution. Injectable intradermally was injected into each marking site using a syringe.

Twenty four hours after the intradermal injection of antiserum, the causative antigen was mixed in the same amount with 1 Evans blue and injected into the rat vein 1 ml each. After injecting the causative antigen into the rat vein, 30 minutes after the cervical dislocation, the rat was slaughtered, and the skin was peeled off to observe the blue spots on the antiserum injection site. The maximum dilution ratio of antiserum with a blue spot of 5 mm in diameter and positive was determined as the PCA antibody value of the antiserum.

Passive skin anaphylaxis test results for mouse antiserum are shown in Table 13 .

Table 13 Results of Passive Skin Anaphylaxis Response Test

group Sensitization Reaction time Mice Diluted voice of sensitized serum 8 16 32 64 128 256 Positive reaction rate I (10 μg / object) Germerin (100 μg / object) 7 14 ------- 0/14 Ⅱ Germerin (100 μg / object) Germerin (100 μg / object) 7 14 ------- 0/14 Ⅲ Grain + FCA ^a (100 μg / individual) Germerin (100 μg / object) 7 14 ------- 0/14 Ⅳ OVA ^b + FCA (0.2 mg / individual) OVA (10 mg / individual) 7 14 -1 4 9--- 14/14 Ⅴ Negative control group (0.2 ml / object) Germerin (100 μg / object) 7 14 ------- 0/14

a: immunoadjuvant

b: albumin

Serum from all groups sensitized with either Kermerin alone or mixed with an adjuvant had an antibody titer of less than 8 and no PCA response was observed. Four were 16-fold and nine were 32-fold PCA antibody titers.

In conclusion, the recombinant germinin of the present invention is unlikely to cause an adverse reaction due to antigenicity in clinical trials and will not cause a specific anaphylactic response when administered to humans.

The present invention was developed to develop a method for dissolving oxygen (STAT) -value fed-batch using an automatic feedback methanol supply control system of recombinant Pichia pastoris using an AOX1 promoter in which expression of an external gene is induced by methanol. The present invention relates to a method for producing a water-soluble recombinant germline having an inhibitory activity with high efficiency and a pharmaceutical composition comprising the same as an active ingredient, wherein the recombinant protein provided by the method is an elastase inhibitor in the same manner as natural germline. As it has the effect of inhibiting the chemotaxis of phagocytic cells, and because it does not induce a humoral immune response in vivo, it can overcome the limitation of the development of the reagent due to the immune response. In addition, since the pharmaceutical composition comprising the recombinant germerin has a wound healing effect, it may be usefully used as a therapeutic agent.

Claims (14)

A method for producing a recombinant germinin using a recombinant Pichia pastoris expression system comprising an AOX1 promoter gene and a germline structural gene in which the expression of an external gene is induced by methanol in a genome. The method of claim 1, wherein the culture of recombinant Pichia pastoris is performed using a dissolved oxygen (DO) -stat fed-batch method applied with an automatic feedback methanol supply control system. The method of claim 2, wherein the DO-stat fed-batch is carried out in four stages: inoculum, batch, pre-induction fed batch and post-induction fed batch. Method for producing a recombinant germinin, characterized in that consisting of) step. 4. The method of claim 3, wherein induction-value culturing step of induction, the oxygen partial pressure of the inlet gas and the methanol feed rate are automatically adjusted by DO values. 5. The method according to claim 4, wherein the oxygen partial pressure control of the inlet gas is controlled by a computer system using a two-gas mixer at the inlet of air and pure oxygen. The method according to claim 4, wherein the automatic regulation is controlled by using the first circulation system and the second circulation system. 7. The recombination system according to claim 6, wherein the first circulation system regulates DO by controlling oxygen transfer rate due to a change in the pure oxygen content of the gas inlet as in the algorithm described in FIG. 13A. Method of preparing the germinin. 8. The DO control according to claim 7, wherein the DO control value is adjusted to a high value of the pure oxygen ratio at the air inlet when the DO value falls below the DO setting value. Method of producing a recombinant germinin, characterized in that the set value is adjusted to low. The method of claim 6, wherein the second circulation system regulates DO by adjusting oxygen uptake rate by changing methanol supply rate as in the algorithm described in FIG. 13B. 10. The method according to claim 9, wherein the DD control is such that the methanol feed rate is adjusted to a low value when the DO value falls below the DO set value, and the methanol feed rate is adjusted to a high value when the DO value rises above the DO set value. Method of making marine. The method according to claim 8 or 10, wherein the DO setting value is set to 40 to 45. Recombinant germinin produced using the manufacturing method of claim 1. A pharmaceutical composition comprising the recombinant germinin of claim 12 as an active ingredient. The pharmaceutical composition of claim 12, wherein the pharmaceutical composition is effective for wound healing when blocking bacterial infection and does not induce a humoral immune response in the body.