Effect of glucose feeding on the glycosylation quality of antibody produced by a human cell line, F2N78, in fed-batch culture.

The human cell line rF2N78 produces an antibody with a high galactosylation ratio which resembles human IgG. However, it has been observed that the aglycosylated antibody starts to appear when glucose is depleted. To determine whether glucose depletion is a main cause for aglycosylation of the antibody, fed-batch cultures of rF2N78 cells were performed using different feeding cocktails (glucose only, nutrient feeding cocktail without glucose, and nutrient feeding cocktail with glucose). In the fed-batch culture with nutrient feeding cocktail without glucose, aglycosylated antibody was produced in a later phase of culture, when glucose was depleted. Approximately 44 % of antibodies produced were aglycosylated at the end of culture. In contrast, aglycosylated antibody was not produced in cultures with glucose feeding. The expression levels of oligosaccharyl transferases determined by Western blot analysis were similar among the cultures, suggesting that aglycosylation of the antibody was not due to altered expression of oligosaccharyl transferases under glucose-deficient conditions. Thus, it is likely that glucose deficiency led to insufficiency of the precursor for glycosylation and induced aglycosylation of the antibody. Taken together, glucose feeding in fed-batch cultures successfully prevented occurrence of aglycosylated antibody during the cultures, confirming that glucose depletion is a main cause for aglycosylation of antibody.

Simultaneous detection of major enteric viruses using a combimatrix microarray.

Various enteric viruses including norovirus, rotavirus, adenovirus, and astrovirus are the major etiological agents of food-borne and water-borne disease outbreaks and frequently cause non-bacterial gastroenteritis worldwide. Sensitive and high-throughput detection methods for these viral pathogens are compulsory for diagnosing viral pathogens and subsequently improving public health. Hence, we developed a sensitive, specific, and high-throughput analytical assay to detect most major enteric viral pathogens using "Combimatrix" platform oligonucleotide probes. In order to detect four different enteric viral pathogens in a sensitive and simultaneous manner, we first developed a multiplex RT-PCR assay targeting partial gene sequences of these viruses with fluorescent labeling for the subsequent microarray. Then, five olignonucleotides specific to each of the four major enteric viruses were selected for the microarray from the oligonulceotide pools targeting the specific genes obtained by multiplex PCR of these viruses. The oligonucleotide microarray was evaluated against stool specimens containing single or mixed viral species. As a result, we demonstrated that the multiplex RT-PCR assay specifically amplified partial sequences of four enteric viruses and the subsequent microarray assay was capable of sensitive and simultaneous detection of those viruses. The developed method could be useful for diagnosing enteric viruses in both clinical and environmental specimens.

cDNA cloning of Korean human norovirus and nucleotidylylation of VPg by norovirus RNA-dependent RNA polymerase.

Norovirus, a member of the Caliciviridae family, is a major causative agent of gastroenteritis worldwide. The cDNA of the entire genome of human norovirus (HuNV) was cloned using the RNA extracted from the stool sample of a Korean patient. The RNA genome consists of 7,559 nucleotides, carries 3 open reading frames (ORFs), 5 and 3 noncoding regions, and a poly(A) tail at the 3 end. Phylogenic analysis of the nucleotide sequence indicated that it belongs to GII.4, the most dominant genogroup. To analyze RNA synthesis and nucleotidylylation of VPg by RNA-dependent RNA polymerase (RdRp), recombinant RdRp and VPg were expressed in Escherichia coli as His-tagged forms. The HuNV RdRp exhibited template and divalent cation-dependent RNA synthesis in vitro. The HuNV RdRp nucleotidylylated HuNV VPg but not murine norovirus (MNV) VPg, whereas MNV RdRp nucleotidylylated both MNV and HuNV VPg more efficiently than HuNV RdRp.

Murine norovirus-1 3Dpol exhibits RNA-dependent RNA polymerase activity and nucleotidylylates on Tyr of the VPg.

We investigated the roles and biochemical properties of recombinant murine norovirus-1 (MNV-1) 3D(pol) in RNA synthesis and virus genome-linked protein (VPg) nucleotidylylation. We therefore expressed VPg and 3D(pol) of MNV-1 in Escherichia coli. MNV-1 3D(pol) exhibited RNA-dependent RNA polymerase (RdRp) activity in vitro with poly(A) RNA as a template and MnCl(2) as a cofactor. MNV-1 3D(pol) demonstrated optimum RNA-synthesis activity at pH 7.4 and 37 degrees C in the absence of a primer. Further, VPg was guanylylated by MNV-1 3D(pol) in the presence of MnCl(2) in a template-independent manner. The guanylylation reaction conducted with VPg substitution mutants (Y26F, Y40F, Y45F and Y117F) and a deletion mutant (Delta117-124) indicated that Tyr(117) was the probable target site of guanylylation. Homopolymeric RNAs did not enhance VPg guanylylation, whereas in vitro-transcribed (-) subgenomic (SG) and (+)SG RNA enhanced VPg guanylylation by 9.2 and 3.2 times, respectively. Within (-)SG RNA, the (-)ORF3 region played a critical role in enhancing VPg guanylylation, suggesting that the MNV-1 ORF3 region of negative-strand RNA contains a cis-acting element that stimulates 3D(pol)-mediated VPg guanylylation.