Production, purification, and characterization of human α1 proteinase inhibitor from Aspergillus niger

✍ Scribed by Liat Chill; Loc Trinh; Parastoo Azadi; Mayumi Ishihara; Roberto Sonon; Elena Karnaukhova; Yakir Ophir; Basil Golding; Joseph Shiloach

• Publisher: John Wiley and Sons
• Year: 2009
• Tongue: English
• Weight: 779 KB
• Volume: 102
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/bit.22099

✦ Synopsis

Abstract

Human alpha one proteinase inhibitor (α1‐PI) was cloned and expressed in Aspergillus niger, filamentious fungus that can grow in defined media and can perform glycosylation. Submerged culture conditions were established using starch as carbon source, 30% dissolved oxygen concentration, pH 7.0 and 28°C. Eight milligrams per liter of active α1‐PI were secreted to the growth media in about 40 h. Controlling the protein proteolysis was found to be an important factor in the production. The effects of various carbon sources, pH and temperature on the production and stability of the protein were tested and the product was purified and characterized. Two molecular weights variants of the recombinant α1‐PI were produced by the fungus; the difference is attributed to the glycosylated part of the molecule. The two glycoproteins were treated with PNGAse F and the released glycans were analyzed by HPAEC, MALDI/TOF‐MS, NSI‐MS^n^, and GC‐MS. The MALDI and NSI‐ full MS spectra of permethylated N‐glycans revealed that the N‐glycans of both variants contain a series of high‐mannose type glycans with 5–20 hexose units. Monosaccharide analysis showed that these were composed of N‐acetylglucos‐amine, mannose, and galactose. Linkage analysis revealed that the galactosyl component was in the furanoic conformation, which was attaching in a terminal non‐reducing position. The Galactofuranose‐containing high‐mannnose type N‐glycans are typical structures, which recently have been found as part of several glycoproteins produced by Aspergillus niger. Biotechnol. Bioeng. 2009; 102: 828–844. © 2008 Wiley Periodicals, Inc.