title: High-level expression of secreted glycoproteins in transformed lepidopteran insect cells using a novel expression vector.
abstract: An expression cassette for continuous high-level expression of secreted glycoproteins by transformed lepidopteran insect cells has been developed as an alternative to baculovirus and mammalian cell expression systems. The expression cassette utilizes the promoter of the silkmoth cytoplasmic actin gene to drive expression from foreign gene sequences, and also contains the ie-1 transactivator gene and the HR3 enhancer region of BmNPV to stimulate gene expression. Using an antibiotic-resistance selection scheme, we have cloned a Bm5 (silkmoth) cell line overexpressing the secreted glycoprotein juvenile hormone esterase (JHE-KK) at levels of 190 mg/L in batch suspension cultures. A baculovirus (AcNPV) expressing the same gene under the control of the p10 promoter of AcNPV produced only 4 mg/L active JHE in static cultures of infected Sf21 cells. A cloned Bm5 cell line overexpressing a soluble isoform of the alpha-subunit of the granulocyte-macrophage colony stimulating factor receptor (solGMRalpha) was also generated and produced five times more solGMRalpha in static cultures than a cloned BHK cell line obtained by transformation with a recombinant expression cassette utilizing the human cytomegalovirus (CMV) enhancer-promoter system. Finally, we show that recombinant protein expression levels in transformed Bm5 cells remain high in serum-free media, that expression is stable even in the absence of antibiotic selection, and that lepidopteran cells other than Bm5 may be used equally efficiently with this new expression cassette for producing recombinant proteins.
authors: Department of Medical Biochemistry, The University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors: P J
authors: PJ
authors: Farrell
authors: M
authors: M
authors: Lu
authors: J
authors: J
authors: Prevost
authors: C
authors: C
authors: Brown
authors: L
authors: L
authors: Behie
authors: K
authors: K
authors: Iatrou
mesh_terms: D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis / Q000235:genetics; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D006224:Cricetinae; D003587:Cytomegalovirus / Q000378:metabolism; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007915:Lepidoptera / Q000378:metabolism* / Q000821:virology; D017924:Nucleopolyhedroviruses / Q000378:metabolism; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D018411:Spodoptera / Q000378:metabolism / Q000821:virology
chemical_list: D006023:Glycoproteins; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D011994:Recombinant Proteins; D002265:Carboxylic Ester Hydrolases; C038003:juvenile hormone esterase