Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells

Philipps, Björn; Rotmann, Daniel; Wicki, Micha; Mayr, Lorenz M.; Forstner, Michael

doi:10.1016/j.pep.2005.03.020

Cited by 33 publications

(28 citation statements)

References 14 publications

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“…In addition to work within SPINE laboratories, much progress has been made elsewhere in the development of the baculovirus system; the use of unified vectors and robotics (Albala et al, 2000), transfection in suspension and deep-well culture of insect cells (Bahia et al, 2005;McCall et al, 2005) and streamlining the overall process of recombinant baculovirus isolation (Phillips et al, 2005) have all contributed to HTP baculovirus expression such that its systematic use, for example for herpesvirus open-reading-frame-encoded proteins, has been described (Gao et al, 2005). Even the most streamlined system for expression screening in baculovirus takes approximately one week longer than a system based on transient expression in mammalian cells.…”

Section: Expression Of Rptp In 293s/gnt1mentioning

confidence: 99%

Eukaryotic expression: developments for structural proteomics

Aricescu

Assenberg

Bill

et al. 2006

Acta Crystallogr D Biol Crystallogr

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The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe (SPINE) consortium have developed and implemented high-throughput (HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast (Pichia pastoris and Saccharomyces cerevisiae), baculovirusinfected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and proteinproduction pipelines are reported. Strategies for coexpression, selenomethionine labelling (in all three eukaryotic systems) and control of glycosylation (for secreted proteins in mammalian cells) are assessed.

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Section: Expression Of Rptp In 293s/gnt1mentioning

confidence: 99%

Eukaryotic expression: developments for structural proteomics

Aricescu

Assenberg

Bill

et al. 2006

Acta Crystallogr D Biol Crystallogr

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“…In this regard, the reporter gene should be expressed under the control of an early or late promoter, one activated much earlier than the polyhedrin promoter, which is a very late promoter. Philipps et al 22) developed a baculovirus transfer vector (pUltraBac1) that contains the EGFP reporter gene driven by the late promoter sequence (pBasic) and the very late polyhedrin promoter, downstream of which the gene of interest can be cloned. In this case, expression of EGFP starts at about 6 h post-infection while expression of the gene of interest starts at about 20 h post-infection.…”

Section: Discussionmentioning

confidence: 99%

Attempts to Express the A1-GMCSF Immunotoxin in the Baculovirus Expression Vector System

Jahanian-Najafabadi

Bouzari

Oloomi

et al. 2012

Bioscience, Biotechnology, and Biochemistry

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“…Park) were grown in LB medium containing appropriate antibiotics. The transfer vector pUltraBac, containing the egfp gene under the control of the late basic protein promoter (P Basic ) was a kind gift from Dr. Felix Freuler (Philipps et al, 2005). The plasmid pGB2Ωinv-hly encoding both invasin and listeriolysin-O was a gift from Dr. Grillot-Courvalin C (Grillot- Courvalin et al, 1998).…”

Section: Methodsmentioning

confidence: 99%

“…The viral titer was determined with the help of the EGFP marker (Cha et al, 1997). Cells expressing EGFP were observed by a Leica TCS NT confocal microscope, and the titers of the baculoviral stocks were calculated according to the TCID 50 (Philipps et al, 2005). The titration results were obtained as the mean ± SD from triplicate assays.…”

Section: Methodsmentioning

confidence: 99%

Expression of recombinant proteins in insect cells by their direct infection with Escherichia coli transformed with baculovirus bacmids

Yu¹,

Hua²,

Hu³

et al. 2014

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Summary. -The baculovirus-insect cell expression system (BES), one of the most popular systems for expression of eukaryotic proteins, was known to have drawbacks such as laborious manipulation of large-size baculovirus bacmids and the transfection procedure. These problems could be eliminated by direct infection of eukaryotic cells with nonpathogenic bacteria harbouring the respective gene -bactofection. However, it was unknown whether this system could be applied to insect cells. Therefore, in this study, the possibility of delivery of enhanced green fluorescent protein (EGFP) gene as a marker into the insect cell lines Sf9 and BmN-SWU1 using the above-mentioned approach with the Bac-to-Bac system was investigated. Using a simple co-incubation of Escherichia coli strains containing recombinant baculovirus bacmids with the EGFP gene and insect cells, it was possible to demonstrate the EGFP expression in these cells and to obtain high-titer recombinant baculoviral stocks. Furthermore, BmN-SWU1 cells proved more susceptible to the used E. coli strains than Sf9 cells. However, the co-expression of invasin and listeriolysin-O, known to enhance the E. coli-mediated gene delivery to mammalian cells, with EGFP, had no effect on insect cells. Summing up, this study proved that a heterologous gene can be efficiently delivered and expressed in insect cells by their simple incubation with non-pathogenic E. coli strains harboring recombinant baculovirus bacmids with the respective gene.Keywords: baculovirus; bacmid; EGFP; Escherichia coli; insect cell * Corresponding authors. E-mail: yxiaolan@163.com, malixin9@ hotmail.com; phone: +86-27-88666349. Abbreviations: AcNPV = Autographa californica nuclear polyhedrosis virus; BES = baculovirus-insect cell expression system; BmNPV = Bombyx mori nuclear polyhedrosis virus; BmN-SWU1 = ovarian cell line of Bombyx mori; EGFP = green fluorescent protein; MOI = multiplicity of infection; p.i. = post infection; sf9 = Spodoptera frugiperda 9; t.o.i. = time of infection

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Time reduction and process optimization of the baculovirus expression system for more efficient recombinant protein production in insect cells

Cited by 33 publications

References 14 publications

Eukaryotic expression: developments for structural proteomics

Eukaryotic expression: developments for structural proteomics

Attempts to Express the A1-GMCSF Immunotoxin in the Baculovirus Expression Vector System

Expression of recombinant proteins in insect cells by their direct infection with Escherichia coli transformed with baculovirus bacmids

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