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

Abstract

Human alpha one proteinase inhibitor (alpha1-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 degrees C. Eight milligrams per liter of active alpha1-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 alpha1-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.

In VitroEvaluation of a Novel mRNA-Based Therapeutic Strategy for the Treatment of Patients Suffering from Alpha-1-Antitrypsin Deficiency

“…However, augmentation therapy is associated with adverse reactions like headache, dizziness, and nausea [42], and annual costs between $60,000 and $150,000 [43]. Recombinant AAT protein has been expressed in different microorganisms [44][45][46], plant cells [47], and human cells [48][49][50], but is linked to disadvantages like nonglycosylation or incorrect glycosylation of the product as well as product contaminations with endotoxins. Plasma-derived AAT, which is purified from human donor blood, is limited and also prone to contamination.…”

Glycosylation Variants of a β-Glucosidase Secreted by a Taiwanese Fungus, Chaetomella raphigera, Exhibit Variant-Specific Catalytic and Biochemical Properties

“…Commonly, N - and O -glycans on fungal glycoproteins are mannan-like structures [43]. Fungal N -glycans contain large mannan chains at non-reducing ends, and some mannoses in the mannan chains could be replaced with galactofuranoses [44]–[47]. On the other hand, fungal O -glycans usually consist of O -mannosyl oligosaccharides.…”

Cytoprotective Effect of Recombinant Human Erythropoietin Produced in Transgenic Tobacco Plants

“…After staining with coomassie stain, EPO bands were excised, and in-gel trypsin digestion was performed as described previously [46]. Glycopeptides were extracted, followed by C18 purification, PNGase A release of N -glycans, and C18 purification of the released glycans.…”