- Email: [email protected]
High-yields heterologous production of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillus oryzae
Journal of Bioscience and Bioengineering VOL. 112 No. 2, 114 – 117, 2011 www.elsevier.com/locate/jbiosc
NOTE
High-yields heterologous production of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillus oryzae Nobuo Yamashita,1,⁎ Yumiko Komori,2 Yoshiyuki Okumura,3 Kei-ichi Uchiya,2 Takeshi Matsui,2 Akira Nishimura,1 Kenji Ogawa,4 and Toshiaki Nikai2 Research & Development Department, Hakutsuru Sake Brewing Co. Ltd, 4-5-5 Sumiyoshiminami-machi, Higashinada-ku, Kobe 658–0041, Japan, 1 Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468–8503, Japan, 2 Department of Validation, Matsuurayakugyo Co. Ltd, 24–21 Enjo-chou, Syowa-ku, Nagoya 466–0054, Japan, 3 and Department of Pulmonary Medicine, Higashi Nagoya National Hospital, 5–101 Umemorizaka, Meito-ku, Nagoya 465–8620, Japan 4 Received 17 January 2011; accepted 30 March 2011 Available online 2 May 2011
AFUEI, an elastase inhibitor produced by Aspergillus fumigatus strongly inhibits the elastolytic activity of A. fumigatus etc. To purify AFUEI, we constructed a strain that overproduces AFUEI by introducing the gene encoding AFUEI (Genbank accession no. AB546725) under control of the amyB promoter into the heterologous host Aspergillus oryzae. A. oryzae TF-4 displayed strong elastase inhibitory activity and produced considerably more AFUEI than that of A. fumigatus. Furthermore, AFUEI could be purified using culture broth and single ultrafiltration (UF) treatment, allowing for the effective production of AFUEI for use in clinical trials. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Elastase inhibitior; Aspergillus fumigatus; Aspergillus oryzae; Heterologous production]
Aspergillosis, a common mycosis occurring mainly in immunocompromised hosts undergoing chemotherapy, is caused by the inhalation of high concentrations of spores from pathogenic Aspergillus species. Because 28% of human lung tissue is composed of elastin, a strong correlation between fungal elastase production and the pathogenesis of aspergillosis has been hypothesized. This hypothesis is of clinical relevance because elastase-producing strains, and in particular Aspergillus fumigatus (1,2), promote a higher rate of death in immunodeficient mice than do non-elastase-producing strains. A. fumigatus has been clearly shown to destroy lung tissue (3,4), and an elastase produced by the opportunistic pathogenic Aspergillus is a confirmed virulence factor in aspergillosis (5). Elastase inhibitors are therefore considered to be an effective therapeutic agent for treatment of aspergillosis. For example, the elastase inhibitor ulinastatin has been administered by intravenous drip infusion concomitantly with antifungal agents to treat patients with pulmonary aspergillosis (6–8). Recently, Okumura et al. isolated a novel elastase inhibitor, AFUEI from A. fumigatus and elucidated its primary structure (9). AFUEI had a very strong inhibitory effect on pathogenic Aspergillus elastases. In addition, AFUEI relatively inhibits human leukocyte elastase suggesting that it is promising agent not only for the treatment of aspergillosis, but also for inflammatory disease. To evaluate the efficiency of AFUEI, clinical trials using a purified protein will be required. However, attempts to purify large
⁎ Corresponding author. Tel.: + 81 78 822 8910; fax: + 81 78 822 8930. E-mail address: [email protected] (N. Yamashita).
amounts of AFUEI have met with little success because this protein is expressed at low levels in A. fumigatus. Therefore, we attempted to produce high yields of AFUEI by overexpressing the AFUEI gene in the heterologous host A. oryzae. DNA was obtained from A. fumigatus AFU-12 (9). We had previously purified AFUEI from A. fumigatus and determined that aa 1–68 were 100% identical to aa 20–87 of the A. fumigatus hypothetical protein AFUA 3 G14940. Based on the known sequence of 3 G14940, we designed primers to amplify the putative AFUEI-encoding gene from A. fumigatus AFU-12. The PCR-amplified fragment was inserted into the expression vector pUSA (10) between the amyB promoter and the terminator to construct pUSAFEI. DNA sequence analysis revealed that the putative AFUEI gene comprised an open reading frame of 371 bp containing two introns (Genbank accession no. AB546725). A. oryzae HL-1036 (sC-)(11), which was derived from industrial A. oryzae HL-1034, was used as the recipient strain after transformation by the protoplast-PEG method (12). Five transformants were isolated and incubated in 20 ml submerged cultures at 30°C for 2 d using Minimal medium (MM)-dextrin (dex)-broth (2% dextrin, 0.6% NaNO3, 0.052% KCl, 0.152% KH2PO4, 0.052% MgSO4·7H2O, and 0.1% trace elements). The elastolytic inhibitory activity of culture supernatants was assayed by the diazo coupling method as previously described (9). Briefly, 4 μl of culture supernatant was mixed with 10 μl of purified A. fumigatus elastase (about 30 ng) at 37°C for 15 min. Next, 180 μl of 50 mM Tris/HCl (pH 7.5) and 4 μl of 50 mM Glutaryl-L-alanyl-L-alanyl-L-proryl-LLeucine-p-nitroanilide (GAAPLNA) (Peptide Institute Inc., Osaka, Japan), with DMSO as the substrate were added and incubated at 37°C
1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2011.03.020
VOL. 112, 2011
NOTE
for 30 min. The reaction was stopped by adding 200 μl of 10% (w/v) trichloroacetic acid. Sodium nitrite (40 μl 0.1%), 40 μl of 0.5% ammonium sulfarate, and 40 μl of 0.1% N-1-naphthylethylenediamine dihydrochloride were then added to the solution, and the amount of p-nitroaniline (pNA) released was quantified by measuring the absorbance at 550 nm. One unit was defined as the amount of AFUEI that reduced pNA release by 1 nmol at 37°C in 1 min. The supernatants of three transformants, TF-3, -4, and −5, showed strong activities: 1764 U/ml [standard deviation (SD) = 7.6%], 2172 U/ml (SD = 8.8%), and 995 U/ml (SD = 7.2%), respectively. No activity was detected in the control strain (TF-C), which was transformed using pUSA at 30°C for 48 h. To examine the profile of the secreted proteins, the supernatants of these four transformants were concentrated 10 times in a vacuum centrifugal evaporator (Sakuma, EC-57CS, Tokyo, Japan) and subjected to SDSPAGE analysis (Fig. 1A) using a e-PAGEL E-T15S slab gel (ATTO, Tokyo, Japan) and Ez Run T (ATTO) as an electrophoretic buffer according to the Laemmli method (13). The gel was stained with Coomassie brilliant blue R-250 to visualize protein bands. The appearace of a band corresponding to AFUEI (7.5 kDa) correlated with the elastase inhibitory activity of the culture supernatant from positive transformants. Asides from Takaamylase at approximately 50 kDa, there was another band that was ovserbed in all AFUEI transformants but not in TF-C. This band is not seen when casamino acid was used as a nitrogen source (Fig. 2A, lanes 3 and 4), suggesting that it is an unknown protein not related to AFUEI. Southern blot analysis using Sal I, a restriction enzyme that will not cleave the open reading frame of AFUEI gene, indicated that TF-3, -4, and −5 each contain multiple copies of the AFUEI gene in their chromosomes (Fig. 1B). Signal intensities measured using a GS-800 densitometer (Bio-Rad, Hercules, CA, USA), indicated a copy number ratio for TF-3, -4, and −5 of approximately 5, 10, and 8, respectively. Therefore, TF-4 appeared to possess the most copies of the AFUEI gene, and the strongest inhibitory activity among the three strains as well. To optimize AFUEI production, the effect of culture medium conditions on AFUEI expression and secretion was examined. Strain TF-4 was cultivated in a submerged culture with five different broth compositions: broth 1: MM-dex; broth 2: MM-dex + 0.5% Yeast extract (YE); broth 3: like broth 1, but with 0.6% casamino acid as the nitrogen source instead of NaNO3; broth 4: broth C + 0.5% YE; and broth 5: DPY
115
(2% dextrin, 1% Polypepton, 0.5% YE)]. SDS-PAGE analysis of culture supernatants demonstrated that TF-4 produced the largest amount of AFUEI (approximately 70.7 μg/ml) in broth 5 (DPY), although there were some additional bands, while broth 2 yielded almost the same level of AFUEI (56.1 μg/ml) but only two additional bands. Hence, it was concluded that it would be best to use broth 2 for the purification of AFUEI (Fig. 2A). After 2 d in MM-dex + 0.5% YE, A. oryzae strain TF-4 produced 15-fold AFUEI than A. fumigatus AFU-12 had produced after 7 d of growth in standard static culture conditions using 1% yeast carbon base (YCB) and 1% casamino acid at 37°C (9). To obtain purified AFUEI, the TF-4 culture supernatant was subjected to an ultrafiltration (UF) treatment (cut-off 30,000, Amicon Ultra, Millipore, Ireland). After concentrating the obtained filtrate 10fold, the purified AFUEI was examined by SDS-PAGE analysis (Fig. 2B). UF treatment clearly removed the two previously observed bands, indicating the recombinant AFUEI polypeptide was purified to apparent homogeneity. In addition, the specific elastase inhibitory activity of the UF-filtrate was nearly 2.9-fold higher than that of the native supernatant, with a yield of approximately 73% after UF. The half maximal inhibitory concentration (IC50 values) of purified AFUEI toward three types of elastases, which were derived from A. fumigatus, human neutrophils (Calbiochem, San Diego, CA, USA), and porcine pancreas (Sigma Chemical Co., St. Louis, MO, USA) were determined. The recombinant AFUEI displayed a strong inhibitory effect on the native elastase of A. fumigatus. Using the same conditions as described above, only low concentrations of both native and recombinant AFUEI (IC50, approximately 2.4 nM) were needed for inhibition, while human neutrophil elastase was also relatively strongly inhibited (IC50, approximately 72 nM) with 50 mM MeOSucAlaAlaProValpNA (Calbiochem) as a substrate. However, under these same condition, no inhibitory activity against the elastase of porcine pancreas was detected. To confirm of the inhibitory effect of recombinant AFUEI in human cells, an in vivo assay was performed using CellTracker™ Green Fluorescent Probe (Lonza Walkersvill, Inc., MD, USA). After a 24-h incubation at 37°C, observations were performed using a fluorescence microscope (Fig. 3). The pre-incubation of recombinant AFUEI and A. fumigatus elastase (Fig. 3D) with approximately 15,000 cells
TFC TF3 TF4 TF5
A
days 1 2
1 2 1 2 1 2
B
TFC TF3 TF4 TF5
kb 23.1
kDa 9.4 6.6
16.9 14.4 10.7 & 8.2 6.2
4.4
AFUEI (7.5kD)
2.3 2.0
FIG. 1. Overexpression of the AFUEI gene in A. oryzae. (A) SDS-PAGE analysis of the 10-fold concentrated supernatant from each transformant after incubation for either 1 or 2 d at the conditions described in the text. (B) Approximately 2 μg of genomic DNA from each transformant was digested with SalI, and a 371 bp fragment encoding the ORF for the AFUEI gene was used as a probe. An ECL labeling and detection system (GE Healthcare UK Ltd., Amersham Place, UK) was used to visualize the DNA bands. The TF-C transformant contains pUSA; the TF-3, -4, and − 5, transformants contain pUAFUEI.
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A
M1 1
J. BIOSCI. BIOENG.,
2
3 4
5
B
M2
M1 1 2 3
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kDa
97 66
97 66 42
42
4 M2
Taka-amylase
30 30 kDa 20 14 AFUEI
20
16.9
14
14.4
AFUEI
10.7, 8.2
kDa 16.9 14.4 10.7, 8.2 6.2
6.2
FIG. 2. Production and purification of AFUEI. (A) Comparison of production using different broths. Lane 1: MM-2% dextrin; lane 2: broth 1 + 0.5% Yeast extract (YE); lane 3: broth 1, with 0.6% casamino acid as a nitrogen source instead of NaNO3; lane 4: broth 3 + 0.5% YE; and lane 5: DPY (2% dextrin, 1% Polypepton, 0.5% YE). The cultures were incubated with shaking (180 rpm) at 30°C for 2 d. (B) Purification of AFUEI using ultrafiltration (UF). Lanes 1 and 3 are culture supernatants from TF-4 after incubation for 48 h in MM-dextrin and MM-dextrin + 0.5% YE, respectively, under the conditions described in the text. Lanes 2 and 4 were loaded with the UF filtrate from lanes 1 and 3, respectively. Lanes M1 and M2 are molecular weight marker.
resulted in a viable cell count almost identical to that of control, to which no elastase and AFUEI was added (Fig. 3A), Upon the addition of only A. fumigatus elastase, almost all of the cell died (Fig. 3B), with the survival rate being lower than 0.5%. These results show that recombinant AFUEI represses the toxicity of A. fumigatus elastase in human cells.
In this study, we successfully cloned and overexpressed the gene encoding A. fumigatus AFUEI in the heterologous host A. oryzae. When combined with a single UF treatment, this approach enabled the effective preparation of purified AFUEI, which appears to be a promising agent for the treatment of aspergillosis or diseases caused by active human elastase.
FIG. 3. Assay of the inhibitory ability of recombinant AFUEI in human cells. Human umbilical vein endothelial cells (HUVEC) and Cell Tracker™ Green Fluorescent Probe were employed for the in vivo assay according to manufacturer's protocol. The viable cell count is indicated on each fluorescence microscope photo. (A) Control (nothing added); (B) addition of A. fumigatus elastase; (C) addition of AFUEI; (D) addition of both A. fumigatus elastase and AFUEI (with pre-incubation at 37°C for 15 min). Initially, approximately 15,000 cells were alive in each culture. A. fumigatus elastase, 1.0 μg; AFUEI, 2.1 μg in 0.2 ml/well of a 96-well micro-titerplate.
VOL. 112, 2011 The authors are grateful to Ms. Haruka Nishimoto for her technical help.
References 1. Frosco, M., Chase, T., Jr., and Machmillan, J. D.: Purification and properties of the elastase from Aspergillus fumigatus, Infect. Immun., 60, 728–734 (1992). 2. Hasegawa, Y., Nikai, T., Yamashita, R., Yoshikawa, Y., Sugihara, H., Ogawa, K., and Mizuno, M.: Isolation and Characterization of elastolytic proteinase from Aspergillus fumigatus, Jpn. J. Med. Mycol., 36, 235–243 (1995). 3. Kothary, M. H., Chase, T., Jr., and Machmillan, J. D.: Correlation of elastase production by some strains of Aspergillus fumigatus with ability to cause pulmonary invasive aspergillosis in mice, Infect. Immun., 43, 320–325 (1984). 4. Reichard, U., Buttner, S., Eiffert, H., Staib, F., and Ruchel, R.: Purification and characterization of an extracellular serine proteinase from Aspergillus fumigatus and its detection in tissue, J. Med. Microbiol., 33, 243–251 (1990). 5. Blanco, J. L., Hontecillas, R., Bouza, E., Blanco, I., Pelaez, T., Munoz, P., Molina, J. P., and Garcia, M. E.: Correlation between the elastase activity index and invasiveness of clinical isolates of Aspergillus fumigatus, J. Clin. Microbiol., 40, 1811–1813 (2002). 6. Hasegawa, Y., Nikai, T., Yoshikawa, Y., Sugihara, H., Ogawa, K., and Takagi, K.: Elastase activity from Aspergillus species. inhibition by ulinastatin, Jpn. J. Med. Mycol., 35, 293–298 (1994).
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7. Maesaki, S., Kohno, S., Tanaka, T., Miyazaki, H., Mitsutake, K., Miyazaki, T., Tomono, K., Kaku, M., Koga, H., and Hara, K.: A case of pulmonary aspergilloma successfully treated with combination therapy of intracavitary injection of amphotericin B and intravenous administration of urinastin, Nihon Kyobu Shikkan Gakkai Zasshi, 31, 1327–1331 (1993) (in Japanese). 8. Ogawa, K.: Treatment of chronic pulmonary Aspergillosis, Kekkaku, 72, 59–64 (1997) (in Japanese). 9. Okumura, Y., Matsui, T., Ogawa, K., Uchiya, K., and Nikai, T.: Biochemical properties and primary structure of elastase inhibitor AFUEI from Aspergillus fumigatus, J. Med. Microbiol., 57, 803–808 (2008). 10. Lee, B. R., Yamada, O., Kitamoto, K., and Takahashi, K.: Cloning, characterization and overexpression of a gene (pdiA) encoding protein disulfide isomerase of Aspergillus oryzae, J. Ferment. Bioeng., 82, 538–543 (1996). 11. Yamashita, N., Sakamoto, K., Yamada, O., Akita, O., and Nishimura, A.: The promoter activity of isovareryl-coA dehydrogenase-encodeing gene (ivdA) from Aspergillus oryzae is strictly repressed by glutamic acid, Biosci. Biotechnol. Biochem., 71, 1561–1563 (2007). 12. Gomi, K., Iimura, Y., and Hara, S.: Integrative transformation of Aspergillus oryzae with a plasmid containing the Aspergillus nidulans argB gene, Agric. Biol. Chem., 51, 2549–2555 (1987). 13. Laemmli, U. K.: Cleavage of structure proteins during the assembly of the head of bacteriophage T4, Nature, 227, 680–685 (1970).
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High-yields heterologous production of the novel Aspergillus fumigatus elastase inhibitor AFUEI in Aspergillus oryzae Nobuo Yamashita,1,⁎ Yumiko Komori,2 Yoshiyuki Okumura,3 Kei-ichi Uchiya,2 Takeshi Matsui,2 Akira Nishimura,1 Kenji Ogawa,4 and Toshiaki Nikai2 Research & Development Department, Hakutsuru Sake Brewing Co. Ltd, 4-5-5 Sumiyoshiminami-machi, Higashinada-ku, Kobe 658–0041, Japan, 1 Department of Microbiology, Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya 468–8503, Japan, 2 Department of Validation, Matsuurayakugyo Co. Ltd, 24–21 Enjo-chou, Syowa-ku, Nagoya 466–0054, Japan, 3 and Department of Pulmonary Medicine, Higashi Nagoya National Hospital, 5–101 Umemorizaka, Meito-ku, Nagoya 465–8620, Japan 4 Received 17 January 2011; accepted 30 March 2011 Available online 2 May 2011
AFUEI, an elastase inhibitor produced by Aspergillus fumigatus strongly inhibits the elastolytic activity of A. fumigatus etc. To purify AFUEI, we constructed a strain that overproduces AFUEI by introducing the gene encoding AFUEI (Genbank accession no. AB546725) under control of the amyB promoter into the heterologous host Aspergillus oryzae. A. oryzae TF-4 displayed strong elastase inhibitory activity and produced considerably more AFUEI than that of A. fumigatus. Furthermore, AFUEI could be purified using culture broth and single ultrafiltration (UF) treatment, allowing for the effective production of AFUEI for use in clinical trials. © 2011, The Society for Biotechnology, Japan. All rights reserved. [Key words: Elastase inhibitior; Aspergillus fumigatus; Aspergillus oryzae; Heterologous production]
Aspergillosis, a common mycosis occurring mainly in immunocompromised hosts undergoing chemotherapy, is caused by the inhalation of high concentrations of spores from pathogenic Aspergillus species. Because 28% of human lung tissue is composed of elastin, a strong correlation between fungal elastase production and the pathogenesis of aspergillosis has been hypothesized. This hypothesis is of clinical relevance because elastase-producing strains, and in particular Aspergillus fumigatus (1,2), promote a higher rate of death in immunodeficient mice than do non-elastase-producing strains. A. fumigatus has been clearly shown to destroy lung tissue (3,4), and an elastase produced by the opportunistic pathogenic Aspergillus is a confirmed virulence factor in aspergillosis (5). Elastase inhibitors are therefore considered to be an effective therapeutic agent for treatment of aspergillosis. For example, the elastase inhibitor ulinastatin has been administered by intravenous drip infusion concomitantly with antifungal agents to treat patients with pulmonary aspergillosis (6–8). Recently, Okumura et al. isolated a novel elastase inhibitor, AFUEI from A. fumigatus and elucidated its primary structure (9). AFUEI had a very strong inhibitory effect on pathogenic Aspergillus elastases. In addition, AFUEI relatively inhibits human leukocyte elastase suggesting that it is promising agent not only for the treatment of aspergillosis, but also for inflammatory disease. To evaluate the efficiency of AFUEI, clinical trials using a purified protein will be required. However, attempts to purify large
⁎ Corresponding author. Tel.: + 81 78 822 8910; fax: + 81 78 822 8930. E-mail address: [email protected] (N. Yamashita).
amounts of AFUEI have met with little success because this protein is expressed at low levels in A. fumigatus. Therefore, we attempted to produce high yields of AFUEI by overexpressing the AFUEI gene in the heterologous host A. oryzae. DNA was obtained from A. fumigatus AFU-12 (9). We had previously purified AFUEI from A. fumigatus and determined that aa 1–68 were 100% identical to aa 20–87 of the A. fumigatus hypothetical protein AFUA 3 G14940. Based on the known sequence of 3 G14940, we designed primers to amplify the putative AFUEI-encoding gene from A. fumigatus AFU-12. The PCR-amplified fragment was inserted into the expression vector pUSA (10) between the amyB promoter and the terminator to construct pUSAFEI. DNA sequence analysis revealed that the putative AFUEI gene comprised an open reading frame of 371 bp containing two introns (Genbank accession no. AB546725). A. oryzae HL-1036 (sC-)(11), which was derived from industrial A. oryzae HL-1034, was used as the recipient strain after transformation by the protoplast-PEG method (12). Five transformants were isolated and incubated in 20 ml submerged cultures at 30°C for 2 d using Minimal medium (MM)-dextrin (dex)-broth (2% dextrin, 0.6% NaNO3, 0.052% KCl, 0.152% KH2PO4, 0.052% MgSO4·7H2O, and 0.1% trace elements). The elastolytic inhibitory activity of culture supernatants was assayed by the diazo coupling method as previously described (9). Briefly, 4 μl of culture supernatant was mixed with 10 μl of purified A. fumigatus elastase (about 30 ng) at 37°C for 15 min. Next, 180 μl of 50 mM Tris/HCl (pH 7.5) and 4 μl of 50 mM Glutaryl-L-alanyl-L-alanyl-L-proryl-LLeucine-p-nitroanilide (GAAPLNA) (Peptide Institute Inc., Osaka, Japan), with DMSO as the substrate were added and incubated at 37°C
1389-1723/$ - see front matter © 2011, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2011.03.020
VOL. 112, 2011
NOTE
for 30 min. The reaction was stopped by adding 200 μl of 10% (w/v) trichloroacetic acid. Sodium nitrite (40 μl 0.1%), 40 μl of 0.5% ammonium sulfarate, and 40 μl of 0.1% N-1-naphthylethylenediamine dihydrochloride were then added to the solution, and the amount of p-nitroaniline (pNA) released was quantified by measuring the absorbance at 550 nm. One unit was defined as the amount of AFUEI that reduced pNA release by 1 nmol at 37°C in 1 min. The supernatants of three transformants, TF-3, -4, and −5, showed strong activities: 1764 U/ml [standard deviation (SD) = 7.6%], 2172 U/ml (SD = 8.8%), and 995 U/ml (SD = 7.2%), respectively. No activity was detected in the control strain (TF-C), which was transformed using pUSA at 30°C for 48 h. To examine the profile of the secreted proteins, the supernatants of these four transformants were concentrated 10 times in a vacuum centrifugal evaporator (Sakuma, EC-57CS, Tokyo, Japan) and subjected to SDSPAGE analysis (Fig. 1A) using a e-PAGEL E-T15S slab gel (ATTO, Tokyo, Japan) and Ez Run T (ATTO) as an electrophoretic buffer according to the Laemmli method (13). The gel was stained with Coomassie brilliant blue R-250 to visualize protein bands. The appearace of a band corresponding to AFUEI (7.5 kDa) correlated with the elastase inhibitory activity of the culture supernatant from positive transformants. Asides from Takaamylase at approximately 50 kDa, there was another band that was ovserbed in all AFUEI transformants but not in TF-C. This band is not seen when casamino acid was used as a nitrogen source (Fig. 2A, lanes 3 and 4), suggesting that it is an unknown protein not related to AFUEI. Southern blot analysis using Sal I, a restriction enzyme that will not cleave the open reading frame of AFUEI gene, indicated that TF-3, -4, and −5 each contain multiple copies of the AFUEI gene in their chromosomes (Fig. 1B). Signal intensities measured using a GS-800 densitometer (Bio-Rad, Hercules, CA, USA), indicated a copy number ratio for TF-3, -4, and −5 of approximately 5, 10, and 8, respectively. Therefore, TF-4 appeared to possess the most copies of the AFUEI gene, and the strongest inhibitory activity among the three strains as well. To optimize AFUEI production, the effect of culture medium conditions on AFUEI expression and secretion was examined. Strain TF-4 was cultivated in a submerged culture with five different broth compositions: broth 1: MM-dex; broth 2: MM-dex + 0.5% Yeast extract (YE); broth 3: like broth 1, but with 0.6% casamino acid as the nitrogen source instead of NaNO3; broth 4: broth C + 0.5% YE; and broth 5: DPY
115
(2% dextrin, 1% Polypepton, 0.5% YE)]. SDS-PAGE analysis of culture supernatants demonstrated that TF-4 produced the largest amount of AFUEI (approximately 70.7 μg/ml) in broth 5 (DPY), although there were some additional bands, while broth 2 yielded almost the same level of AFUEI (56.1 μg/ml) but only two additional bands. Hence, it was concluded that it would be best to use broth 2 for the purification of AFUEI (Fig. 2A). After 2 d in MM-dex + 0.5% YE, A. oryzae strain TF-4 produced 15-fold AFUEI than A. fumigatus AFU-12 had produced after 7 d of growth in standard static culture conditions using 1% yeast carbon base (YCB) and 1% casamino acid at 37°C (9). To obtain purified AFUEI, the TF-4 culture supernatant was subjected to an ultrafiltration (UF) treatment (cut-off 30,000, Amicon Ultra, Millipore, Ireland). After concentrating the obtained filtrate 10fold, the purified AFUEI was examined by SDS-PAGE analysis (Fig. 2B). UF treatment clearly removed the two previously observed bands, indicating the recombinant AFUEI polypeptide was purified to apparent homogeneity. In addition, the specific elastase inhibitory activity of the UF-filtrate was nearly 2.9-fold higher than that of the native supernatant, with a yield of approximately 73% after UF. The half maximal inhibitory concentration (IC50 values) of purified AFUEI toward three types of elastases, which were derived from A. fumigatus, human neutrophils (Calbiochem, San Diego, CA, USA), and porcine pancreas (Sigma Chemical Co., St. Louis, MO, USA) were determined. The recombinant AFUEI displayed a strong inhibitory effect on the native elastase of A. fumigatus. Using the same conditions as described above, only low concentrations of both native and recombinant AFUEI (IC50, approximately 2.4 nM) were needed for inhibition, while human neutrophil elastase was also relatively strongly inhibited (IC50, approximately 72 nM) with 50 mM MeOSucAlaAlaProValpNA (Calbiochem) as a substrate. However, under these same condition, no inhibitory activity against the elastase of porcine pancreas was detected. To confirm of the inhibitory effect of recombinant AFUEI in human cells, an in vivo assay was performed using CellTracker™ Green Fluorescent Probe (Lonza Walkersvill, Inc., MD, USA). After a 24-h incubation at 37°C, observations were performed using a fluorescence microscope (Fig. 3). The pre-incubation of recombinant AFUEI and A. fumigatus elastase (Fig. 3D) with approximately 15,000 cells
TFC TF3 TF4 TF5
A
days 1 2
1 2 1 2 1 2
B
TFC TF3 TF4 TF5
kb 23.1
kDa 9.4 6.6
16.9 14.4 10.7 & 8.2 6.2
4.4
AFUEI (7.5kD)
2.3 2.0
FIG. 1. Overexpression of the AFUEI gene in A. oryzae. (A) SDS-PAGE analysis of the 10-fold concentrated supernatant from each transformant after incubation for either 1 or 2 d at the conditions described in the text. (B) Approximately 2 μg of genomic DNA from each transformant was digested with SalI, and a 371 bp fragment encoding the ORF for the AFUEI gene was used as a probe. An ECL labeling and detection system (GE Healthcare UK Ltd., Amersham Place, UK) was used to visualize the DNA bands. The TF-C transformant contains pUSA; the TF-3, -4, and − 5, transformants contain pUAFUEI.
116
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A
M1 1
J. BIOSCI. BIOENG.,
2
3 4
5
B
M2
M1 1 2 3
kDa
kDa
97 66
97 66 42
42
4 M2
Taka-amylase
30 30 kDa 20 14 AFUEI
20
16.9
14
14.4
AFUEI
10.7, 8.2
kDa 16.9 14.4 10.7, 8.2 6.2
6.2
FIG. 2. Production and purification of AFUEI. (A) Comparison of production using different broths. Lane 1: MM-2% dextrin; lane 2: broth 1 + 0.5% Yeast extract (YE); lane 3: broth 1, with 0.6% casamino acid as a nitrogen source instead of NaNO3; lane 4: broth 3 + 0.5% YE; and lane 5: DPY (2% dextrin, 1% Polypepton, 0.5% YE). The cultures were incubated with shaking (180 rpm) at 30°C for 2 d. (B) Purification of AFUEI using ultrafiltration (UF). Lanes 1 and 3 are culture supernatants from TF-4 after incubation for 48 h in MM-dextrin and MM-dextrin + 0.5% YE, respectively, under the conditions described in the text. Lanes 2 and 4 were loaded with the UF filtrate from lanes 1 and 3, respectively. Lanes M1 and M2 are molecular weight marker.
resulted in a viable cell count almost identical to that of control, to which no elastase and AFUEI was added (Fig. 3A), Upon the addition of only A. fumigatus elastase, almost all of the cell died (Fig. 3B), with the survival rate being lower than 0.5%. These results show that recombinant AFUEI represses the toxicity of A. fumigatus elastase in human cells.
In this study, we successfully cloned and overexpressed the gene encoding A. fumigatus AFUEI in the heterologous host A. oryzae. When combined with a single UF treatment, this approach enabled the effective preparation of purified AFUEI, which appears to be a promising agent for the treatment of aspergillosis or diseases caused by active human elastase.
FIG. 3. Assay of the inhibitory ability of recombinant AFUEI in human cells. Human umbilical vein endothelial cells (HUVEC) and Cell Tracker™ Green Fluorescent Probe were employed for the in vivo assay according to manufacturer's protocol. The viable cell count is indicated on each fluorescence microscope photo. (A) Control (nothing added); (B) addition of A. fumigatus elastase; (C) addition of AFUEI; (D) addition of both A. fumigatus elastase and AFUEI (with pre-incubation at 37°C for 15 min). Initially, approximately 15,000 cells were alive in each culture. A. fumigatus elastase, 1.0 μg; AFUEI, 2.1 μg in 0.2 ml/well of a 96-well micro-titerplate.
VOL. 112, 2011 The authors are grateful to Ms. Haruka Nishimoto for her technical help.
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