Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/81—Protease inhibitors
  • C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• FIG. 2 represents a comparison of elastase-inhibiting activity between the selected clones producing Guamerin, #3- 32 and #12-44,
• FIG. 9 represents time courses of methanol feeding rate after induction
• FIG. 12 is a photograph of western blot analysis which indicates a comparison of intracellular and extracellular form of the recombinant Guamerin after induction, where
• FIG. 13b represents the balanced DO-stat algorithm of the present invention for control of DO by methanol feeding rate
• FIG. 14 is a graph indicating absorbance and elastase- inhibiting activity of the recombinant Guamerin purified by using hydrophobic interaction chromatography (A) and reverse phase chromatography (B) ,
• FIG. 15 is a graph indicating the result of C18 reverse- phase HPLC analysis of fractions obtained from each purification step for recombinant Guamerin,
• FIG. 16 is a graph of binding stoichiometry and inhibition kinetics, wherein binding stoichiometry indicates that the recombinant Guamerin binds to porcine pancreatic elastase (PPP) with an equimolar ratio,
• PPP porcine pancreatic elastase
• FIG. 17 is a graph indicating stability of the recombinant Guamerin at various pHs (A) and temperature (B) ,
• FIG. 19 is a photograph observed by naked eyes, which shows that wound healing is quicker when the recombinant Guamerin containing fibrin glue is applied to bacterial infection-excluded wound than when a control are applied
• FIG. 20 is a photograph observed under microscope, which shows that decrease of degeneration in connective tissue is more when the recombinant Guamerin containing fibrin glue is applied to bacterial infection-excluded wound than when a control is applied.
• the structural gene In large-scale expression using a microorganism, the structural gene must be genetically stable throughout the production process including several passages of seed culture.
• the generation number elapsed for preparation of cells working in expression step from MCB (master cell bank) averages about 5 to 7 in the system.
• MCB master cell bank
• large-scale fermentation undergoes more generation to enlarge the volume of culture, its generation number does not exceed 20.
• the stability of structural gene up to generation number of 70 indicates that the industrial-scale fermentation is possible.
• AOXl promoter In P. pastoris expression system, AOXl promoter is used (Buckholz et al . , 1991; Cregg et al . , 1993).
• methanol is firstly oxidized by alcohol oxidase, and used as a carbon source. Alcohol oxidase is undetected in cells cultured on carbon sources such as glucose, glycerol or ethanol, but constitutes up to 30% of total soluble proteins in cells grown on methanol as carbon sources.
• carbon sources such as glucose, glycerol or ethanol
• foreign genes are maintained in an expression-off mode on non- methanolic carbon sources, then efficiently switched on by shifting to media containing methanol.
• methanol is consumed as a carbon source and an inducer simultaneously.
• methanol In the growth of P. pastoris , methanol can pass through the oxidative pathway only if there are sufficient oxygen molecules. However, the oxygen deficiency inhibits the further oxidation of methanol, which resulted in accumulation of methanol in the culture broth. Eventually, toxicity of accumulated methanol could exert fatal damage to both cell growth and the recombinant Guamerin expression. Thus, methanol concentration and dissolved oxygen level are very important control factors in the post-induction fed-batch phase of P. pastoris expression process. Control schemes of methanol concentration and dissolved oxygen in expression process, are interdependent in a complicated manner and cannot be performed efficiently without consideration of this interdependence . In conventional fed-batch culture for the cultivation of P.
• DO level was kept high by manual manipulation of agitation and oxygen mixing in inlet air stream. Both variables were controlled independently. However, it has been found that the manual manipulation is not suitable for methanol feeding due to occurrence of overfeeding or underfeeding.
• DO-stat fed-batch culture process using two interrelated loops is a improved control method for the prevention of methanol overfeeding or underfeeding.
• the present inventors adopt automatic feedback methanol feeding rate control system for more delicate methanol feeding rate control.
• This control method protects cells from both oxygen deficiency and excess methanol supply.
• the control method uses computer system to control efficiently the supply process.
• the application of the balanced DO-stat strategy for high cell culture of microorganisms can be realized by another simpler structure in which DO is controlled directly by control of OUR and OTR.
• the present inventors introduce the concept of DO-stat which is controlled by simultaneous control of the OTR and OUR.
• cell density of P. pastoris reaches 140 g/L at the start point of the methanol feeding post-induction fed-batch period.
• oxygen demand is so large that OTR can not sustain OUR even at the maximum agitation speed.
• the change of pure oxygen mixing ratio in the inlet air stream is introduced to control the OTR for the balanced DO-stat fed batch process.
• the present inventors also control OUR by manipulation of the methanol feeding rate.
• the proposed control structure was designed to consist of two interrelated loops. DO is controlled at a narrow intervals of set point (40-50%) .
• the first loop controls DO via manipulating OTR which is resulted from the variation of the portion of pure oxygen gas in the inlet gas stream (see FIG. 13a) .
• DO value is dropped below the lower limit of DO set point (D0 low )
• DO value rises above upper limit of DO set point (DO up )
• the set point of the pure oxygen ratio in the inlet air stream is adjusted to lower value.
• the second loop controls the DO value via manipulating OUR by variation of methanol feeding rate (see FIG 13b) .
• inoculum cultured in YPD media containing glycerol is inoculated into fermentor containing minimal salt medium.
• Fermentor pH is maintained at 5.1 by automatic addition of 15% (v/v) NH 4 OH and 30% (v/v) H 3 P0 4 , respectively.
• the dissolved oxygen level is maintained above 30% saturation by automatic adjustment of the agitation speed and oxygen partial pressure of inlet gas.
• the oxygen partial pressure of inlet gas is controlled by mixing air and pure oxygen in inlet gas stream by Two Gas Mixers controlled by computer.
• the temperature of the fermentor is maintained at 30°C.
• the cells are allowed to grow about 15 hours in the batch mode until all glycerol are exhausted (dry cell weight > 25 g/L) .
• DO-stat fed-batch is performed using the glycerol medium containing glycerol and yeast trace metal.
• feed pump is turned on, and when DO level falls below the set point, the feed pump stops. This procedure is repeated by checking DO level every 30 seconds using computer aided BioCommand Software (New Brunswick Scientific Co.).
• induction of recombinant Guamerin production medium containing methanol and yeast trace metal solution is fed, and feeding rate is increased by the stepwise mode. At early induction stage, a low feeding rate is maintained for 4 hours to activate the expression promoter.
• the expression level of recombinant Guamerin produced by the improved DO-stat fed-batch culture of the present invention is 40% higher than the result from the conventional fed-batch, in which medium is manually fed.
• the recombinant Guamerin is secreted into the medium thoroughly immediately after expression, which reveals that DO-stat fed-batch culture of the present invention was efficient for the recombinant Guamerin production.
• the present invention provides pharmaceutical composition containing the recombinant Guamerin as an active ingredient for wound healing.
• the present inventors purify the recombinant Guamerin from the recombinant P. pastoris and analyze its biochemical characteristics.
• Elastase-inhibiting activity was measured by using porcine pancreatic elastase and N-succinyl-Ala-Ala-Ala-p- nitroanilide were used as the enzyme and its substrate, respectively.
• One inhibition unit (iu) is defined as the amount of the inhibitor suppressing the liberation of 1 ⁇ mole of p-nitroanilide per min from N-succinyl-Ala-Ala- Ala-p-nitroanilide under the given conditions.
• the inhibition constant (K was determined based on the tight- binding kinetics and calculated using the following equation .
• ⁇ is the fractional activity of elastase not bound to the inhibitor
• E 0 and I 0 are the initial concentrations of the enzyme and inhibitor, respectively.
• the pH and temperature stability of the recombinant Guamerin were examined. It was found that more than 90% of the inhibiting activity was retained even when the recombinant Guamerin was incubated for 24 hours at pHs ranging from 1 to 12. The recombinant Guamerin also exhibited an excellent temperature stability, retaining more than 90% of its activity after incubation for 6 hours at an elevated temperature such as 90°C.
• Guamerin conferred by its many disulfide bonds creats its pH and temperature stability, which means that the recombinant Guamerin of the present invention is expressed with a correct conformation.
• the recombinant Guamerin of the present invention has enough therapeutic potential as a new drug candidate for wound healing.
• the present inventors performed animal experiments using mice.
• Guamerin a strong elastase inhibitor, has inhibiting effect on chemostasis of phagocytes, anti- inflammatory effect may be expected. Whereas, since Guamerin inhibits primary phagocytosis of phagocytes at early stage of infection in case of bacterial infection, the inflammation can be become worse. In the present invention, when the recombinant Guamerin added to fibrin glue was applied under the situation which bacterial infection was excluded, the Guamerin showed quicker effect for wound healing than control .
• compositions containing the Guamerin can be administered via a parental route, and be used in the form of general pharmaceutical formulation.
• the therapeutical agents can be administered via a parental route with several formulations .
• formulations generally-used fillers, extenders, binders, wetting agents, disintegrating agents, diluents such as surfactant or excipients may be used.
• Formulations for a parental administration include sterilized aqueous solutions, water-insoluble excipients, suspensions, emulsions and suppositories.
• water-insoluble excipients and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable esters such as ethyloleate.
• the Guamerin of the present invention was treated with fibrin glue in a preferred embodiment of the present invention.
• the effective dose of the recombinant Guamerin for wound healing is preferably 1 ng to 100 mg per 1 cm 2 of wound area .
• Example 1 Identification of genetic stability of P. pastoris expression system and high-cell-density culture using it
• the structural gene for Guamerin obtained from cDNA library of whole leech by PCR was fused with ⁇ - factor leader sequence containing KEX2 cleavage site to construct expression vector, pGS29A, and thereafter transformed into P. pastoris GS115, a his4 strain to construct expression strain using P. pastoris (Korean patent No.: 98-27344) .
• the gene was inserted by a single crossover between the his4 locus in the chromosome and the HIS4 marker in the vector.
• Fig. 1 represents the expression vector, pGS29A.
• GS115 P. pastoris host strain, and the transformants were cultured in YPD medium, complex medium (1% yeast extract, 2% peptone, 2% glucose), and MD medium (1.34% YNB, 4X10 _5 % biotin, 2% glucose), respectively.
• P. pastoris GS115 methylotrophic yeast was transformed with pGS29A by sphero flast method. The transformants were streaked on MD and cultured at 30°C until colonies were formed. Several hundreds of colonies selected primarily were inoculated into MGY liquid medium (1.34% YNB, 4X10 "5 % biotin, 1% glycerol) and cultured at 30°C, 250 rpm.
• the medium was replaced with MM liquid medium (1.34% YNB, 4X10 " 5 % biotin, 0.5% methanol) which induces expression of the protein in the transformants, and cultured at 30°C, 250 rpm for 70 hours.
• the culture broth was centrifuged and the supernatant was separated to primarily examine and compare elastase-inhibiting activity of the recombinant Guamerin.
• Porcine pancreatic elastase PPE, sigma
• N- Succinyl-Ala-Ala-Ala-p-nitroanilide were used as an enzyme and its substrate for measuring elastase-inhibiting activity, respectively.
• the reaction was performed under the buffer condition, 0.05 M Tris-HCl (pH 8.0).
• 1 volume of buffer solution containing PPE 0.1 unit
• Two clones, #3-32 and #12-44, with highest elastase- inhibiting activity were selected.
• expression by methanol was induced in the following experiment.
• the two clones were inoculated into minimal medium and cultured for 15 hours.
• the cell pellets recovered by centrifugation were resuspended in minimal medium containing 0.5% methanol instead of glycerol.
• Methanol was repeatedly supplied every 24 hours to maintain the methanol concentration of 0.5% for three days.
• the elastase-inhibiting activity was compared between #3-32 and #12-44 by the above method.
• MCB MCB was cultured in YPD medium for 16 hours. 1:1 mixture of culture broth and 50% glycerol was made in cryo-tube (Corning, USA) and MCB was stored at -70°C. To assure the culture homogeneity, 360 colonies were isolated from one vial of MCB and tested the phenotype for auxotrophic selection marker, his4. Colonies isolated from non-selection media containing L-histidine were picked and then transferred to selection media containing no L- histidine. All colonies picked were formed new colonies in selection media after incubation at 37°C for 18 hours (FIG. 3) . It could be concluded that all colonies in MCB were harbored homogeneously the expression cassette in its genome .
• the isolated genomic DNA of P. pastoris was analyzed by Southern blotting after treatment of restriction enzymes. DNA isolation from the recombinant P. pastoris was performed using Easy-DNA kit (Invitrogen Co., U.S.A.). Southern blotting was also performed using the various combinations of restriction enzymes and probes like Bglll/Guamerin, EcoRI /HIS and EcoPI/Guamerin. The results of Southern blotting was analyzed based on the principle of homologous recombination at his4 locus of chromosomal DNA. Probe against the structural gene of Guamerin was prepared using random prime labeling module (Gene ImageTM, Amersham International, UK) .
• HIS4 and Guamerin were amplified by PCR using pGS29A vector as a template. Random primers were bound to the amplified JJJS4 and Guamerin, a template. Fluorescein-11-dUTPs were integrated into the probes formed during random labelling reaction by klenow fragment of DNA polymerase I . The probes containing Fluorescein-11-dUTPs were bound to anti-fluorescein conjugated to alkaline phosphatase (AP) . AP degraded dioxetane, thereby generating chemiluminescence . The resulting chemiluminescent signals were exposed on film.
• AP alkaline phosphatase
• Table 1 showed the expected size of restricted DNA fragments detected by Southern blotting when expression cassettes were integrated by the manner of homologous recombination at his locus. As shown in Table 1, multiple gene insertion could be detected by the results of Southern blotting. To confirm the number of the integrated cassettes, Bglll and HIS4 probe were used. As a result, fragments of 3.7, 4.7 and 5.8 kb were detected with the ratio of intensity of 1:1:1 (FIG. 4). This result means that two copies of expression cassette were inserted at the his4 locus of the P. pastoris genome. Fig. 5 showed expected restriction map when two copies of expression cassette were integrated. Besides the combination as mentioned above, when Bglll /Guamerin, EcoRI /His4 and EcoRI /Guamerin were used for restriction analysis, restriction maps were the same as that shown in Fig. 6 (FIG. 6) .
• a single colony from Guamerin-producing P. pastoris was inoculated into 50 mL of YPD medium and then overnight culture was performed in shaking incubator at 30°C, 150 rpm. This flask culture was used as an inoculum for the new flask culture and this relay culture was performed repeatedly until generation number reached about 70.
• 10 mL of culture broth was withdrawn from every relay flask culture just before the culture was used as an inoculum. Total DNA of the cell was treated with Sail, electrophoresed on 0.6% agarose gel and transferred to nylon membrane for blotting. Probe against the structural gene of Guamerin was prepared using random prime labeling module (Gene ImagesTM, Amersham International pic, UK) .
• DNA frgment containing the structural gene of Guamerin was stable up to about generation number of 70.
• the integrated structural gene must be stable throughout the production process including several passages of seed culture.
• the generation number being elapsed for preparation of WCB (working cell bank) strain which expresses recombinant protein, from MCB, original strain, averages about 5-7 in the culture system of the present invention.
• the volume of culture was enlarged for large-scale fermentation, its generation number does not exceed 20.
• the stability of structural gene up to generation number of 70 could be enough for industrial utilization.
• the relay culture test demonstrated that the genetic stability of the structural gene of Guamerin was stable beyond the normal generation number required for industrial production process.
• the fed-batch culture was performed consecutively, in which the medium feeding was controlled by DO-stat mode with 50% air-saturation as a set point.
• the feed medium was composed of 50% (w/v) glycerol and 12 mL/L of yeast trace metal solution.
• the medium feeding by DO-stat mode was stopped and then the culture was starved for 0.5 hour to consume the residual glycerol completely.
• the medium containing 50% of methanol and 12 mL/L of yeast trace metal solution was fed to induce the Guamerin production and its feeding rate was increased manually from 22 to 48 mL/h by the stepwise mode for 40 hours. As shown in Fig.
• Example 2 Production method of the recombinant Guamerin using the improved P. pastoris expression system
• inoculum In the improved DO-stat fed-batch experiment for the recombinant Guamerin production, growth was divided into four phase: inoculum, batch, pre-induction fed-batch and post-induction fed-batch.
• the inocula were started by adding 100 ⁇ L of a thawed glycerol stock solution to a flask containing 100 mL of YPD ' media (l%(w/v) glucose, 1% (w/v) yeast extract, 2% (w/v) peptone). The flask was incubated for 20 hours in shaker at 30°C, 150 rpm.
• the flask contents were then added to 2.5 L Bioflow III fermentor (New Brunswick Scientific) containing minimal salt medium (glycerol, 40 g/L; H 3 P0 4 , 27 mL; CaS0 4 , 0.9 g/L; K 2 S0 4 , 18.0 g/L; MgS0 4 , 10.24 g/L; KOH, 4.13 g/L; NH 4 OH, 30 mL/L; yeast trace metal solution, 4.4 mL/L).
• minimal salt medium glycerol, 40 g/L; H 3 P0 4 , 27 mL; CaS0 4 , 0.9 g/L; K 2 S0 4 , 18.0 g/L; MgS0 4 , 10.24 g/L; KOH, 4.13 g/L; NH 4 OH, 30 mL/L; yeast trace metal solution, 4.4 mL/L).
• Yeast trace metal solution was composed of as follows; CuS0 4 -5H 2 0, 6.0 g/L; KI, 0.09 g/L; MnS0 4 -H 2 0, 3.0 g/L; NaMo-H 2 0, 24.0 g/L; H 3 B0 4 , 0.02 g/L; CoCl,, 0.5 g/L; ZnCl 2 , 20.0 g/L; FeS0 4 -H 2 0, 65.0 g/L; Biotin, 0.2 g/L; H 2 S0 4 , 20.0 mL/L. Fermentation vessel pH was maintained at 5.1 by automatic addition of 15% (v/v) NH 4 OH and 30% (v/v) H 3 P0 4 , respectively.
• the dissolved oxygen level was maintained above 30% saturation by automatic adjustment of the agitation speed and oxygen partial pressure of inlet gas.
• the oxygen partial pressure of inlet gas was controlled by mixing of air and pure oxygen in inlet gas stream using Two Gas Mixers controlled by computer (New Brunswick Scientific, Edison) .
• the temperature of fermentation vessels was maintained at 30°C.
• the cells were allowed to grow for 15 hours in the batch mode until all glycerol was exhausted (dry cell weight>25 g/L) .
• DO-stat fed-batch was performed for 13 hours with the glycerol medium containing of 80% (w/v) glycerol and 12 mL of yeast trace metal.
• the first three phase were the same as the conventional fed-batch culture.
• DO was controlled at a narrow intervals of a set point (40-45%) .
• DO value was dropped below the lower limit of DO set point (D0 low )
• DO value rose above upper limit of DO set point (DO up )
• DO up the pure oxygen ratio in the inlet air stream was adjusted to lower value.
• DO value was dropped below the lower limit of DO set point
• a pentium 133 MHz based PC system was used for on-line data measurement, calculation and control of the present invention.
• the computer was linked with fermentor through an RS422 interface.
• the collected data including dissolved oxygen, pH, temperature, agitation speed and nutrient pump speed were analyzed and controlled by BioCommand software.
• cell density was determined by Dry Cell Weight (DCW) measurement. To determine cell density, 10 mL of culture broth was centrifuged at 9000 rpm and the cell pellet was washed with phosphate buffered saline (PBS) and again centrifuged. This procedure was repeated twice. Finally, the cell pellet was resuspended in distilled water and dried using electronic moisture analyzer (Sartorius MA 40, Sartorius, Germany) .
• DCW Dry Cell Weight
• Glycerol and methanol concentrations were measured by gas chromatography (HP5890 series II, Hewlett Packard, USA). 6 ft OV-17 WHP 100/120 19001A-B12 and 6 ft 10% degs whp 100/120 19001a-L12 columns were used for glycerol and methanol measurement, respectively.
• the result from the method of the present invention was compared with the result from the fed-batch of which feeding rate was controlled manually.
• the proposed two control loops were operated simultaneously and well suited for on-line computer application. Indeed, after initial tuning, there was no need to change the parameters.
• Culture profile of the improved DO-stat was shown in Fig. 10. Twenty four hours after induction, the expression level of the recombinant Guamerin was 450 U/L which was 40% higher than the final result from the conventional fed-batch, which was shown in Fig. 11. In this case, western blot analysis showed that little amount of the recombinant Guamerin was remained in the cell during the production stage (FIG. 12) . That is, the recombinant Guamerin was secreted into the medium thoroughly immediately after expression. This revealed that our fermentation process maintained the well- constructed secretion machinery of the recombinant P. pastoris .
• the production yield was increased due to the well-constructed culture system of the recombinant P. pastoris using DO-stat fed-batch culture for producing the recombinant Guamerin of the present invention.
• Example 3 Purifica ion of the recombinant Guamerin .
• the produced recombinant Guamerin was purified.
• HIC hydrophobic interaction chromatography
• ammonium sulfate was first added to the supernatant from the culture broth containing the expressed recombinant Guamerin, to a final concentration of 1.0 M.
• the solution was then applied to an HIC column (Econo, 5 cm X 13 cm, BioRad, USA) which was packed with phenyl sepharose 6FF (Amersham Pharmacia Biotech, Sweden) and pre-equilibrated with 0.05 M Tris-HCl (pH 8.0) containing 1.0 M ammonium sulfate.
• the protein was eluted with a linear gradient of 1.5 L of 1.0 to 0.0 M ammonium sulfate with a flow rate of 50 mL/h.
• the fractions containing the recombinant Guamerin were pooled, filtered by a 0.2 ⁇ m-fliter and then applied to the column (C8-100 10SP, 2.5 cm X 10 cm, Amicon) which was pre-equilibrated with 2% (v/v) 2-propanol for reverse phase chromatography
• the elastase-inhibiting activity of the recombinant Guamerin of the present invention was 603 iu/g and increased up to 9.3 fold after purification. This shows that the portion of recombinant Guamerin in the culture medium was 11% of the total protein.
• the inhibition kinetics showed that the recombinant Guamerin was a reversible-equimolar tight- binding inhibitor of porcine pancreatic elastase with Ki of 38 nm.
• binding stoichiometry and inhibition kinetics shown in Fig. 16 indicates that the recombinant Guamerin binds to porcine pancreatic elastase with an equimolar ratio.
• N-terminal amino acid sequence analysis was performed using an automatic peptide sequencer (Model 492, ProciseTM, Perkin-Elmer) .
• amino acid composition analysis the recombinant Guamerin was hydrolyzed in 6 N
• mice were bled at the end of the first, second and fourth week after the first immunization.
• the second immunization was performed at the end of the second week after the first immunization.
• the antibody titers for each group were quantified using the enzyme-linked immunoassay (ELISA) method (Tijssen, P., Elsevier Science Publishers B.V., The Netherlands, 1985).
• the titer was defined as the dilution factor of a sample giving a positive reaction as determined by the cut-off value of the mean optical density plus three times the standard deviation of the negative control sera of the mice.
• A PBS
• B Freund' s adjuvant
• C Guamerin in PBS
• D D:
• mice were used for each sample
• the recombinant Guamerin of the present invention does not cause humoral immune responses, it may be able to overcome the immunogenic problem which has been the major obstacle in the development of a new drug.
• the acute toxcity test for the recombinant Guamerin of the present invention was performed to develop a new drug using the recombinant Guamerin.
• As antigenicity tests active systemic anaphylaxis test (ASA test) and passive cutaneous anaphylaxis test (PCA test) were performed.
• the recombinant Guamerin was freeze-dried using 2- propanol/water as a solvent, the resulting recombinant Guamerin in the form of white powder was resuspended in sterilized saline and was used for acute toxicity test.
• ovalbumin sigma
• a positive control in antigenicity test ovalbumin (sigma) dissolved in sterilized saline was used as a mixture (1:1) with Freund' s complete adjuvant (Gibco) .
• a sterilized saline, a solvent was used as a negative control.
• SPF specific pathogen free mouse (17-24 g) was used.
• SPF specific pathogen free mouse (17-24 g) was used.
• polycarbonate breeding box temperature, 23 ⁇ 3°C; relative humidity, 50+10%; exhaustion, 10-12 times/h; light and darkness of a fluorescent light, 12 hours cycle; intensity of illumination, 150-160 lux
• the experimental animals ate solid feed (Shin Chon Feed Co.) and tap water as a beverage.
• Body weight was measured for the animals which were determined based up their health during the acclimation period, the animals having body weights verging on average body weight were selected, and grouping was performed using a random method. Differences between average body weights among groups were identified by performing a statistical verification using an ANOVA (one way analysis of variance) verification.
• ANOVA one way analysis of variance
• Untreated control was administered once via intravenous route. After the recombinant Guamerin was diluted to the concentration suitable to each test group using distilled water on the date of the test, it was administered microvenously in mice by 1 ml syringe. Doses were calculated based on the body weights immediately before venous administration.
• the clinical signs for all the test animals were detected every hour from 1 hour to 6 hours after the administration on the date of test, and observed more than once per day at a fixed time from the first day to the fourteenth day after the administration. After the administration, the animals were observed as to their changes of general state, toxicosis expression, death and the signs which were able to appear by Guamerin. Body weights of 11 animals used for the test were measured on the date of administration, the forth day, seventh day, twelfth day and the fourteenth day.
• Body weights of the alive animals after the test were measured before autopsy, and observed in detail whether or not their external appearances and internal organs were damaged after autopsy.
• the ANOVA was used for statistical analysis, and significances of the experimental groups with control group were verified at the levels of P ⁇ 0.05 and P ⁇ 0.01.
• the 50% lethal dose (LD 50 ) of Guamerin will be more than 1,000 mg/kg on intravenous route .
• NGF no gross finding, percent of no gross finding
• a group was composed of 7 guinea pigs and all the guinea pigs were divided into 5 groups. 10 ⁇ g/animal of the recombinant Guamerin was administered in group 1, 100 ⁇ g/animal of the recombinant Guamerin in group 2, 100 ⁇ g/animal plus Freund' s complete adjuvant (FCA) in group 3, ovalbumin 0.2 mg/animal plus FCA in group 4 and vehicle in group 5.
• FCA Freund' s complete adjuvant
• FCA ovalbumin 0.2 mg/animal plus FCA in group 4 and vehicle in group 5.
• the substances were administered subcutaneously for 9 times in 3 weeks (3 times/week) and in mixed administration groups, the substances were administered subcutaneously for 3 times in 3 weeks (once/week) .
• the experimental substances dissolved in sterilized saline were used for the test.
• antigens (Guamerin 100 ⁇ g/animal or ovalbumin 10 mg/animal) were administered via tarsal vein of guinea pigs. For 30 min after induction, systemic signs were observed to identify whether or not anaphylactic shocks were detected, and the results of the test were evaluated according to the criteria of Table 10.
• the substances were administered subcutaneously for 9 times in 3 weeks (3 times/week) and in mixed administration groups, the substances were administered subcutaneously for 3 times in 3 weeks (once/week) .
• the experimental groups, dose, times and route were shown in Table 13.
• blood was extracted from mouse orbital venous plexus and serum was separated.
• fur at the back of rats was removed as wide as possible using clipper, left and right side of the back was marked using a red magic pen.
• 100 ⁇ L of mouse anti-serum which was diluted for six times from 8 fold by stepwise mode using sterilized saline, was administered intradermally at the marked site using 1 mL syringe of 26 1/2 G.
• Table 13 showed the results of passive cutaneous anaphylaxis test against mouse anti-serum.
• the recombinant Guamerin of the present invention has rare possibility of occurring side effects due to antigenicity in clinical tests, and is estimated not to induce anaphylaxis responses when it is administered in human.
• the present invention relates to a method for efficiently preparing a soluble recombinant Guamerin having elastase-inhibiting activity in recombinant P. pastoris, and pharmaceutical compositions containing the recombinant
• Guamerin as a therapeutical agent for wound healing.
• DO dissolved oxygen
• the recombinant Guamerin provided by the method of the present invention inhibits chemotaxis of phagocytes as an elastase inhibitor like native Guamerin. Since the recombinant Guamerin does not induce humoral immunological responses in vivo, it is able to overcome immunological problem which has been the limit in the development of a new drug.
• the pharmaceutical compositions containing the recombinant Guamerin are effective for wound healing, they can be used as a therapeutic agent for elastase-associated diseases.

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