Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger.
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.
Chill
Liat
L
Biotechnology Core Laboratory, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 14A Room 170, 9000 Rockville Pike, Bethesda, Maryland 20892, USA.
Trinh
Loc
L
Azadi
Parastoo
P
Ishihara
Mayumi
M
Sonon
Roberto
R
Karnaukhova
Elena
E
Ophir
Yakir
Y
Golding
Basil
B
Shiloach
Joseph
J
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000235:genetics / Q000378:metabolism*; D018533:Biomass; D019149:Bioreactors; D002236:Carbohydrate Conformation; D050260:Carbohydrate Metabolism; D002241:Carbohydrates / Q000737:chemistry; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000737:chemistry; D055550:Protein Stability; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D000515:alpha 1-Antitrypsin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D002241:Carbohydrates; D011134:Polysaccharides; D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase