Sung-Wan Kim

Changes in cellular secretory processing during baculovirus infection

In the baculovirus expression vector system (BEVS), secretory green fluorescent protein (sGFP) transcripts were expressed from day 2 to day 5 post-infection (p.i.), while transcripts of the endoplasmic reticulum (ER) molecular chaperone Bombyx mori protein disulfide isomerase (bPDI) were measurable in mock cells and in cells at day 1 p.i. The GFP was expressed from day 3 to day 5 p.i. whereas the levels of two ER chaperone proteins, bPDI and calnexin, decreased from day 3 p.i. and were not detected from day 4 p.i. These findings suggest that the rate-limited expression of ER molecular chaperones is strongly associated with the maximal expression of exogenous proteins in BEVS.

Galatosylation and sialylation of mammalian glycoproteins produced by baculovirus-madiated gene expression in insect cells.

The baculovirus expression vector system (BEVS) is used extensively for the production of proteins from exogenous cDNAs. However, BEVS is not ideal for pharmaceutical production of glycoproteins owing to the properties of the N-glycans in the expressed products and that insect cells lack several of the enzymes required for mammalian-type N-glycan synthesis. This study describes the effective mammalian-like production of glycoproteins, such as β-1,4-galactosyltransferase and α-2,6-sialyltransferase, in the insect cell line Sf9.

A Powerful Ubiquitous Activity of Bombyx mori Heat Shock Protein 70 Promoter

For stable germline transformation, the promoter of Bombyx mori cytoplasmic actin gene (BmA3) has been used for ubiquitous expression of transgenes. So far, no strong promoter is available for ubiquitous expression in B. mori, excluding BmA3 promoter. To identify more powerful promoter than previously reported BmA3 promoter, we isolated 9 clones that show stronger signal compared to BmA3 by a dot blot hybridization. Among these 9 clones, we focused on one clone which has high amino acid homology (94%) with heat shock protein 70 gene of Trichoplusia ni. This clone, named bHsp70 was ubiquitously expressed in all tissues and developmental stage of 5th instar B. mori larvae, and stimulated by thermal and ER stress. As result of promoter assay using dual luciferase assay system, we found the highest transcription activity region (−1003/+147) in the 5′-flanking region of bHsp70 gene, which has about 264 fold more intensive promoter activity than BmA3 promoter. Transcription activity of bHsp70 promoter under heat shock condition (42°C, 4 hr) was increased over 2 fold than normal condition. In bHsp70 promoter, there are an ER stress response element (ERSE) and a heat shock element (HSE). Accordingly, we suppose that the bHsp70 promoter may be used for conditional expression of heterogonous proteins under thermal or ER stress. Moreover, the bHsp70 promoter was normally regulated in Bm5, Sf9, and S2 cells. Therefore, we suggest that bHsp70 promoter may be used more powerful and effectively for transgene expression in various insects containing B. mori as a universal promoter.

Identification of the unfolded protein response (UPR)-related genes from Bombyx mori cell lines by a subtractive hybridization approach

The baculovirus expression vector system (BEVS) is one of the powerful insect cell systems for heterologous protein expression. However, over-expression of heterologous proteins in this system sometimes results in protein misfolding and aggregation because of insufficient levels of folding catalysts. In previous study using the differential screening (DS) method, we isolated only 40 differentially expressed genes after treatment with tunicamycin, an unfolded protein response (UPR) inducer. To isolate more protein folding catalysts from insect, we performed suppressive subtractive hybridization (SSH) with untreated and tunicamycin-treated Bm5 cell lines in this study. We could isolate 366 differentially expressed clones by SSH method and produced expressed sequence tags (ESTs). ESTs included the UPR pathway-related genes involved in protein folding, including heat shock proteins, molecular chaperones, foldases, as well as glycosylation and secretory pathway related genes. Identification of the tunicamycin responsive genes using SSH provides more information about the UPR-related genes in insect cells, and will facilitate modifications of the protein folding pathway in the ER to improve heterologous protein expression.

Bombyx mori Protein Disulfide Isomerase (bPDI) Protects Sf9 Cells from Endoplasmic Reticulum (ER) Stress

In the previous our study, a cDNA that encodes protein disulfide isomerase from Bombyx mori (bPDI) was isolated and characterized. bPDI has an open reading frame of 494 amino acids contained two PDI-typical thioredoxin active site of WCGHCK and ER (endoplasmic reticulum) retention signal of the KDEL motif at its C-terminal. Recent studies have demonstrated that misfolded proteins are accumulated in many diseases including Alzheimers, goiter, emphysema, and prion infections. bPDI was over-expressed or knock-downed in Sf9 cells to study the relationship between bPDI expression and protections against protein misfolding. bPDI gene was cloned in insect expression vector pIZT/V5-His for over-expression and bPDI double-stranded RNA (dsRNA) was generated for knock-down. Over-expression of bPDI significantly improved survival rate, but bPDI dsRNA transfection significantly reduced survival rate after 48 hours exposure. In mock-transfected or wild-type cells had no significant effect. The results support the view that bPDI is one of the important intracellular components for cell protect mechanism, especially, against ER stress such as protein misfolding.