The development of stably transformed lepidopteran insect cell technology for both the expression of recombinant proteins and the generation of baculovirus artificial chromosomes.

摘要

Our present work has focused on the development of transformed insect cell expression technology with characteristics that are superior to other expression systems, particularly the baculovirus expression system.;An expression cassette and protocols for continuous, high-level expression of secreted glycoproteins from transformed insect cell lines was developed. The cassette utilizes silkmoth cytoplasmic actin gene promoter to drive foreign gene expression, and also contains the ie-1 transactivator gene and the HR3 enhancer region of the Bombyx mori nuclear polyhedrosis virus to stimulate gene expression. Using an antibiotic resistance selection scheme, a cloned Bm5 cell line (silkmoth) transformed with the expression cassette containing the secreted glycoprotein juvenile hormone esterase (JHE) as a reporter gene, produced 210 mug/mL in stirred suspension culture, and 150 mug/mL in serum-free medium in static culture. The baculovirus expression system (AcNPV infected Sf21 cells) could only produce 4 mug/mL active JHE in static cultures. This cell line exhibited stable recombinant protein expression for over 4 months, and lepidopteran insect cells other than Bm5 cells were shown to be equally efficient for producing recombinant proteins with this expression cassette.;For the expression of intracellular proteins from genetically engineered organisms, we generated DNA coding for a secretion module---a fusion protein that contains JHE at the N-terminus. This resulted in the efficient secretion of two intracellular proteins from transfected insect cells. An intra-protein histidine tag allowed purification of the fusion protein, and an intra-protein enteropeptidase cleavage site was recognized for liberation of the intracellular protein from the secretion module.;Collaborations were established with other research groups to express proteins in insect cells that could not be produced efficiently in other protein expression systems. Four secreted proteins [human tissue plasminogen activator (t-PA), human granulocyte-macrophage colony-stimulating factor (GM-CSF), a soluble isoform of the alpha subunit of the human GM-CSF receptor (solGMralpha) and a non-glycosylated form of bovine transferrin (ngbTF)], one G-protein coupled membrane receptor [rat protease activated receptor 2 (rPAR-2)], two ion exchangers [native bovine retinal rod Na+-Ca2+K + exchanger (bNCKX) and a modified bNCKX (bNCKXdd)], and a secreted intracellular protein [Bombyx mori chorion factor 1 (BmCF1)] were successfully expressed. Comparisons of expression levels or biological activity with other expression systems were found to favor the use transformed insect cells for recombinant protein expression.;Stably transformed insect cell lines were used to create b&barbelow;aculov&barbelow;irus a&barbelow;rtificial c&barbelow;hromosomes (BVACs). Our approach was to inactivate a single baculovirus gene rendering an infectious, yet harmless, self-replicating extra-chromosomal entity that can carry useful genes into lepidopteran cells. Rescuing insect cell lines were generated to make BVACs and infectious BVAC inocula. However, the successful generation of pure BVACs was hampered by recombination events where the virulence was regained by the BVACs from the rescuing cell line. (Abstract shortened by UMI.).