Print Email Facebook Twitter Expression of human α1-proteinase inhibitor in Aspergillus niger Title Expression of human α1-proteinase inhibitor in Aspergillus niger Author Karnaukhova, E. Ophir, Y. Trinh, L. Dalal, N. Punt, P.J. Golding, B. Shiloach, J. TNO Kwaliteit van Leven Publication year 2007 Abstract 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. Subject BiologyBiotechnologyalpha 1 antitrypsinglucan 1,4 alpha glucosidaseglucan 1,4 alpha glucosidase 2recombinant alpha 1 antitrypsintrypsin inhibitorarticleAspergillus nigerbiotechnological productioncontrolled studyenzyme linked immunosorbent assayglycosylationmatrix assisted laser desorption ionization time of flight mass spectrometrymolecular weightnonhumannucleotide sequencepolyacrylamide gel electrophoresisprotein degradationprotein determinationprotein expressionprotein synthesisWestern blottingAspergillus nigerFungi To reference this document use: http://resolver.tudelft.nl/uuid:f72731e6-6c6b-45ec-a984-442b879f8d79 DOI https://doi.org/10.1186/1475-2859-6-34 TNO identifier 240253 ISSN 1475-2859 Source Microbial Cell Factories, 6 Article number No.: 34 Document type article Files To receive the publication files, please send an e-mail request to TNO Library.