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Expression of human α1-proteinase inhibitor in Aspergillus niger

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Author: Karnaukhova, E. · Ophir, Y. · Trinh, L. · Dalal, N. · Punt, P.J. · Golding, B. · Shiloach, J.
Institution: TNO Kwaliteit van Leven
Source:Microbial Cell Factories, 6
Identifier: 240253
doi: doi:10.1186/1475-2859-6-34
Article number: No.: 34
Keywords: Biology · Biotechnology · alpha 1 antitrypsin · glucan 1,4 alpha glucosidase · glucan 1,4 alpha glucosidase 2 · recombinant alpha 1 antitrypsin · trypsin inhibitor · article · Aspergillus niger · biotechnological production · controlled study · enzyme linked immunosorbent assay · glycosylation · matrix assisted laser desorption ionization time of flight mass spectrometry · molecular weight · nonhuman · nucleotide sequence · polyacrylamide gel electrophoresis · protein degradation · protein determination · protein expression · protein synthesis · Western blotting · Aspergillus niger · Fungi


Background: Human α1-proteinase inhibitor (α1-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, α1-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 α1-PI (r-α1-PI) could provide an attractive alternative. Although r-α1-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. Results: We have explored the possibility of expressing the gene for human α1-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more αmammalian-likeα glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of α1-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 α1-PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-α1-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-α1-PI was similar to that of the pd-α1-PI. In vitro stability of the r-α1-PI from A. niger was tested in comparison with pd-α1-PI reference and non-glycosylated human r-α1-PI from E. coli. Conclusion: We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for α1-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for α1-PI in A. niger was successfully achieved to produce the secreted mature human r-α1-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. © 2007 Karnaukhova et al; licensee BioMed Central Ltd.