Ftouhi Paquin N, Tarentino A L, Plummer T H
Division of Molecular Medicine, Wadsworth Center, New York State Department of Health, Empire State Plaza, Albany, New York, 12201-0509, USA.
Protein Expr Purif. 1998 Nov;14(2):302-8. doi: 10.1006/prep.1998.0966.
Peptide-N4-(N-acetyl-beta-d-glucosaminyl asparagine amidase) from Aspergillus tubigensis (PNGase At) was expressed in baculovirus-infected insect cells. The recombinant PNGase At was secreted and purified to homogeneity with a yield of 9.5 mg per liter of infected cell medium. Recombinant PNGase At migrated upon SDS-PAGE as a single-chain protein with a molecular mass of 78 kDa. This contrasts with the native Aspergillus enzyme which is "nicked" and migrates as two subunits each with a molecular weight about 43 kDa. Quantitation of total sugar by phenol-sulfuric acid suggests that the enzyme expressed in baculovirus-infected insect cells was substituted with 8-10 chains of carbohydrate of which 75% was released by Endoglycosidase F1. ESI-MS analysis of the oligosaccharides released from the recombinant PNGase At revealed similarity in the number of glycosylated residues but a significant difference in their composition, when compared to the carbohydrates of the native PNGase At. Despite differences in the primary structure and in the composition of glycan residues, the recombinant enzyme had the same specific activity as the native enzyme.
来自曲霉（PNGase At）的肽 - N4 -（N - 乙酰 - β - D - 葡糖胺基天冬酰胺酶）在杆状病毒感染的昆虫细胞中表达。重组PNGase At被分泌并纯化至同质，每升感染细胞培养基的产量为9.5毫克。重组PNGase At在SDS - PAGE上迁移时表现为一条分子量为78 kDa的单链蛋白。这与天然曲霉酶不同，天然曲霉酶是“切口”的，以两个亚基迁移，每个亚基分子量约为43 kDa。通过苯酚 - 硫酸法对总糖进行定量分析表明，在杆状病毒感染的昆虫细胞中表达的该酶被8 - 10条碳水化合物链取代，其中75%可被内切糖苷酶F1释放。对从重组PNGase At释放的寡糖进行电喷雾电离质谱（ESI - MS）分析发现，与天然PNGase At的碳水化合物相比，糖基化残基的数量相似，但组成有显著差异。尽管一级结构和聚糖残基组成存在差异，但重组酶与天然酶具有相同的比活性。
