Enhanced recombinant protein production and differential expression of molecular chaperones in sf-caspase-1-repressed stable cells after baculovirus infection

Abstract

BackgroundThere are few studies that have examined the potential of RNA inference (RNAi) to increase protein production in the baculovirus expression vector system (BEVS). Spodoptera frugiperda (fall armyworm) (Sf)-caspase-1-repressed stable cells exhibit resistance to apoptosis and enhancement of recombinant protein production. However, the mechanism of recombinant protein augmentation in baculovirus-infected Caspase-repressed insect cells has not been elucidated.ResultsIn the current study, we utilized RNAi-mediated Sf-caspase-1-repressed stable cells to clarify how the resistance to apoptosis can enhance both intracellular (firefly luciferase) and extracellular (secreted alkaline phosphatase [SEAP]) recombinant protein production in BEVS. Since the expression of molecular chaperones is strongly associated with the maximal production of exogenous proteins in BEVS, the differential expression of molecular chaperones in baculovirus-infected stable cells was also analyzed in this study.ConclusionThe data indicated that the retention of expression of molecular chaperones in baculovirus-infected Sf-caspase-1-repressed stable cells give the higher recombinant protein accumulation.

Development of a baculovirus vector carrying a small hairpin RNA for suppression of sf-caspase-1 expression and improvement of recombinant protein production

“…Long dsRNAs have been shown to be able to be expressed and interfere with the transcription of caspase-1 in stably transfected Sf and Tn cells [ 31 – 33 ], and stable Bm cells expressing shRNAs have also been made to suppress the Bm-caspase-1 [ 30 ]. Suppression of the caspase-1 expression improved the production of recombinant proteins in these insect cells with BEVS.…”

“…Here, we detected higher GFP fluorescence produced by an infection using a baculovirus expressing Sf-caspase-1 shRNA1 from 2 to 4 dpi, but it seemed that the accumulated GFP level was equivalent to the control by 4 dpi. It was reported that the enhanced production of exogenous proteins in BEVS was strongly associated with the retention of molecular chaperone expression in baculovirus-infected Sf-caspase-1 repressed stable cells [ 31 ]. Therefore, we speculated that the expression of chaperones in late baculovirus infections could benefit GFP folding and contribute to the stronger fluorescence intensity.…”

“…Blocking the activity of caspases by RNA interference have been used to improve protein production in mammalian cells [ 28 , 29 ]. In BEVS, several reports have established stable insect cell lines expressing short-hairpin RNAs (shRNA) [ 30 ] or long double stranded RNA (dsRNA) that can be processed by Dicer into siRNAs of 21-23 bp [ 31 – 33 ] to suppress the expression of caspase-1 and detected the improvement of protein production.…”

Establishment of a melanogenesis regulation assay system using a fluorescent protein reporter combined with the promoters for the melanogenesis-related genes in human melanoma cells

“…The transfected cells were named as MeWo/pMITF-EGFP, MeWo/pTyr-EGFP and MeWo/pDct-EGFP stable cells [20]. The genomic DNAs of transfected cells were checked by polymerase chain reaction (PCR) using the primers in Table 1.…”

Curcumin enhances the production of major structural components of elastic fibers, elastin, and fibrillin-1, in normal human fibroblast cells

“…The kinetic assay was performed by reading the absorbance at 405 nm at regular intervals over a 10 min period using an ELISA microplate reader (BioTek, Seattle, WA, USA). 16) RT-PCR analysis.…”