Abstract
BackgroundThe insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production.ResultsSeventy cell lines were established by single-cell cloning from a primary culture of cells derived from eggs of the black witch moth (Ascalapha odorata; Lepidoptera, Noctuidae). Among 8 rapidly growing lines, cell line 38 (Ao38) was selected for further analysis, based on susceptibility to AcMNPV infection and production of secreted alkaline phosphatase (SEAP) from a baculovirus expression vector. In comparisons with low-passage High Five (BTI-Tn-5B1-4) cells, infected Ao38 cells produced β-galactosidase and SEAP at levels higher (153% and 150%, respectively) than those measured from High Five cells. Analysis of N-glycans of SEAP produced in Ao38 cells revealed two N-glycosylation sites and glycosylation patterns similar to those reported for High Five and Sf9 cells. Glycopeptide isoforms consisted of pauci- or oligomannose, with and without fucose on N-acetylglucosamine(s) linked to asparagine residues. Estimates of Ao38 cell volume suggest that Ao38 cells are approximately 2.5× larger than Sf9 cells but only approximately 74% of the size of High Five cells. Ao38 cells were highly susceptible to AcMNPV infection, similar to infectivity of Sf9 cells. Production of infectious AcMNPV budded virions from Ao38 cells peaked at approximately 4.5 × 107 IU/ml, exceeding that from High Five cells while lower than that from Sf9 cells. Ao38 cells grew rapidly in stationary culture with a population doubling time of 20.2 hr, and Ao38 cells were readily adapted to serum-free medium (Sf-900III) and to a suspension culture system. Analysis of Ao38 and a parental Ascalapha odorata cell line indicated that these lines were free of the alphanodavirus that was recently identified as an adventitious agent in High Five cell lines.ConclusionsAo38 cells represent a highly productive new insect cell line that will be useful for heterologous protein expression and other applications in biotechnology.
Highlights
The insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production
Desirable characteristics of an insect cell line include rapid growth, adaptation to and rapid growth in largescale suspension cultures, growth in serum-free media, and susceptibility to infection by AcMNPV and/or other expression vectors
The titres determined on Ao38 and reference High Five cells were 0.95 (+/- 0.10) × 107 and 2.90 (+/- 0.88) × 107 IU/ml, respectively. These results indicated that Ao38 cells were similar to, or slightly more susceptible to AcMNPV infection than Sf9 cells
Summary
The insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production. Insect cell lines are essential for basic studies of insect viruses and arboviruses and represent critical components in the baculovirus expression vector system. In addition insect cell lines have been used for studies of immunological, toxicological, and hormonal responses [1]. Desirable characteristics of an insect cell line include rapid growth (cell division times of ≤ 24 h), adaptation to and rapid growth in largescale suspension cultures, growth in serum-free media, and susceptibility to infection by AcMNPV and/or other expression vectors. An additional factor in the utility of an insect cell line is the possible presence of adventitious agents. Cell lines that are free of adventitious agents are more highly desirable since industrial production of human therapeutic proteins or vaccine candidates must adhere to rigorous specifications related to purity and content
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