Hideki Yamaji

The clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system is a widely used genome editing tool, but its application for the development of stably transformed lepidopteran insect cells for recombinant protein production has been limited. In the present study, we applied the CRISPR-Cas9 system to the targeted transgene knock-in to efficiently develop recombinant insect cells. A double-strand break (DSB) in a target site of the genome of Trichoplusia ni BTI-TN-5B1-4 (High Five) cells was detected by T7 endonuclease assay after the cells were transfected with a plasmid for genome editing by employing a U6-4 promoter derived from T. ni to drive guide RNA (gRNA). Genomic DNA analysis via PCR confirmed the integration of a transgene into a target site of the High Five cell genome. The length of the homology arm affected the genome editing efficiency, and the use of a 500-bp version proved to be optimal. Via the co-transfection of High Five cells with gRNA-Cas9 plasmid and a donor vector harboring a reporter gene and the neomycin resistance gene, 21 clones of stably transformed cells were established. In 19 of those clones a site-specific knock-in of the reporter gene at the target site was confirmed, which indicates that an exogenous gene can be efficiently integrated into a target site of the High Five cell genome via the CRISPR-Cas9 system, though transcriptional hot spots in the genome should be identified to establish highly productive recombinant cells.

Influenza vaccines have long been manufactured in embryonated chicken eggs. This method has some problems such as a long production period (about 6 months) and use of large amounts of infectious pathogens. Recently, the production of recombinant subunit vaccines using the baculovirus–insect cell system has been extensively investigated. In this system, viral immunodominant components can be produced more rapidly and in a larger scale than in the conventional egg-based process. However, continuous production is virtually impossible because infection of recombinant baculovirus results in the death of host insect cells. In the present study, we established stably transformed insect cells that secreted influenza virus-like particles (VLPs) consisting of hemagglutinin (HA), the major protective antigen of influenza A virus, and matrix protein 1 (M1), another structural protein of the virus. Hemagglutination assay and transmission electron microscopy (TEM) suggested that HA produced by recombinant insect cells kept the hemagglutination activity and the morphology of the VLPs was similar to that of wild type influenza virus particles.

Insect cells have recently proven to be an excellent platform for the high-level production of functional recombinant proteins. Autophagy is an important mechanism that promotes cell survival by eliminating damaged organelles and protein aggregates, and it also may influence recombinant protein production. In the present study, we compared the effects that autophagy inducers rapamycin, everolimus, and lithium chloride exert on recombinant lepidopteran insect cells that secrete an engineered antibody molecule. Compared with nontreatment, treatment with either rapamycin or everolimus prolonged cell growth to allow high cell density, improved viability in the declining phase, and then increased the yield of secreted antibodies. These positive effects appeared to be induced via autophagy since autophagosomes were clearly detected, particularly in cells treated with rapamycin or everolimus. Unlike rapamycin, another autophagy inducer, FK506, was ineffective in insect cells. The addition of an appropriate autophagy inducer may be effective in increasing the productivity of recombinant proteins in insect cells.

Monoclonal antibodies and antibody fragments are widely used in therapeutics and diagnoses. While mammalian cells serve as the host cells for antibody production, insect cells can produce large quantities of secretory antibodies in serum-free suspension cultures. The effects of lithium on the processes of autophagy and apoptosis in mammalian cells are well chronicled. In the present study, stably transformed insect cells, which produce an engineered antibody molecule, were cultured with lithium chloride in a serum-free medium. Treatment with lithium chloride induced autophagy and apoptosis in recombinant insect cells and led to increases in the yields of secreted antibodies.

The aromatic compound p-hydroxybenzoate (PHBA) is an important material with multiple applications, including as a building block of liquid crystal polymers in chemical industries. The cytochrome P450 (CYP) enzymes are beneficial monooxygenases for the synthesis of chemicals, and CYP53A15 from fungus Cochliobolus lunatus is capable of executing the hydroxylation from benzoate to PHBA. Here, we constructed a system for the bioconversion of benzoate to PHBA in Escherichia coli cells coexpressing CYP53A15 and human NADPH-P450 oxidoreductase (CPR) genes as a redox partner. For suitable coexpression of CYP53A15 and CPR, we originally constructed five plasmids in which we replaced the N-terminal transmembrane region of CYP53A15 with a portion of the N-terminus of various mammalian P450s. PHBA productivity was the greatest when CYP53A15 expression was induced at 20°C in 2×YT medium in host E. coli strain ΔgcvR transformed with an N-terminal transmembrane region of rabbit CYP2C3. By optimizing each reaction condition (reaction temperature, substrate concentration, reaction time, and E. coli cell concentration), we achieved 90% whole-cell conversion of benzoate. Our data demonstrate that the described novel E. coli bioconversion system is a more efficient tool for PHBA production from benzoate than the previously described yeast system.