Expression of human α1-proteinase inhibitor in Aspergillus niger

Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda,Maryland, 20892 USA. .
Microbial Cell Factories (Impact Factor: 4.22). 02/2007; 6(1):34. DOI: 10.1186/1475-2859-6-34
Source: PubMed


Human alpha1-proteinase inhibitor (alpha1-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, alpha1-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant alpha1-PI (r-alpha1-PI) could provide an attractive alternative. Although r-alpha1-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation.
We have explored the possibility of expressing the gene for human alpha1-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of alpha1-PI with a strongly expressed, secreted leader protein (glucoamylase G2), separated by dibasic processing site (N-V-I-S-K-R) that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and alpha1-PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-alpha1-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis further confirmed that molecular mass of the r-alpha1-PI was similar to that of the pd-alpha1-PI. In vitro stability of the r-alpha1-PI from A. niger was tested in comparison with pd-alpha1-PI reference and non-glycosylated human r-alpha1-PI from E. coli.
We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for alpha1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for alpha1-PI in A. niger was successfully achieved to produce the secreted mature human r-alpha1-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth.

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    • "adhA human: granulocyte macrophage colony stimulating factor GM-CSF (Davies 1994) amdS Boophilus microplus (cattle tick): cell surface glycoprotein (Turnbull et al. 1989, 1990) E. coli: enterotoxin subunit B amy (taka) Thermomyces lanuginosus: lipase (Prathumpai et al. 2004b) aphA human: interferon-α-2 (MacRae et al. 1993) glaA human: interleukin-6, interferon-α-2, mucus proteinase inhibitor, antigen-binding (Fab') fragment, humanized IgG1(kappa) antibodies (Archer et al. 1990) porcine: pancreatic phospholipase A2, prochymosin (Broekhuijsen et al. 1993) hen: egg-white lysozyme (HEWL) (Carrez et al. 1990) Thaumatococcus daniellii: Thaumatin (Conesa et al. 2000) Phanerochaete chrysosporium: lignin peroxidase H8 and manganese peroxidase H4 (Contreras et al. 1991) Mucor miehei: triglyceride lipase, aspartyl protease (Frenken et al. 1998) Agaricus meleagris: pyranose dehydrogenase (Gordon et al. 2000) Aequoria victoria: green fluorescent protein (Jeenes et al. 1993) Clostridium thermocellum: dockerin (Levasseur et al. 2004) (Mikosch et al. 1996) (Pisanelli et al. 2010) (Roberts et al. 1992) (van den Hondel et al. 1991) (Ward et al. 2004) gpdA human: α1-proteinase inhibitor (antitrypsin), interleukin-6 (Archer et al. 1990) hen: egg-white lysozyme HEWL) (Bohlin et al. 2006) Pycnoporus cinnabarinus: laccase (Contreras et al. 1991) Trametes versicolor: laccase (Record et al. 2002) E. coli: β-galactosidase, β-glucuronidase (Karnaukhova et al. 2007) sucA/pkiA Aequoria victoria: green fluorescent protein (gfp) (Roth and Dersch 2010) Thermobifida fusca: T. fusca hydrolase (tfh) (Yuan et al. 2006) A. awamori amyA/amyB bovine: prochymosin (Korman et al. 1990) exlA human: interleukin-6 (Gouka et al. 1996b, 1997) Cyamopsis tetragonoloba: α-galactosidase Thermomyces lanuginose: lipase Escherichia coli: β-glucuronidase gam/glaA human: lactoferrin, (Dunn-Coleman et al. 1991) bovine: prochymosin B (Gouka et al. 1997) Thaumatococcus daniellii: Thaumatin (Faus et al. 1998) (Ward et al. 1990) gdhA Thaumatococcus daniellii: Thaumatin (Moralejo et al. 1999) gdhA (A. nidulans) Thaumatococcus daniellii: Thaumatin (Moralejo et al. 1999) A. oryzae amyA (taka) human: lactoferrin, lysozyme, single-chain variable region fragment (scFv) anti lysozyme construct (Gomi et al. 2000) calf: chymosin (Todaka et al. 2009) Reticulitermes speratus: (termite) endoglucanase (Huge-Jensen et al. 1989) Mucor miehei: triglyceride lipase, aspartyl protease (Nakajima et al. 2006) Thermomyces lanuginose: lipase (Sotiriadis et al. 2001) Curculigo latifolia: sweet fruit protein Neoculin (van den Hondel et al. 1991) (Yaver et al. 2000) (Ward et al. 1992a) glaA calf: chymosin (Tsuchiya et al. 1992) fungal transcriptional control regions is crucial for the construction of efficient expression vectors. "
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