Use of juvenile hormone esterase as a novel reporter enzyme in the baculovirus expression system.
Juvenile hormone esterase (JHE) has a number of characteristics favorable for use as a reporter enzyme. It is extremely stable under a variety of adverse conditions including in organic solvent. JHE is easily detected by a rapid and sensitive colorimetric assay, and detection is facilitated by export of the enzyme from the cell. Its use is illustrated in the baculovirus expression system by promoter studies and evaluation of culture conditions necessary for optimal production of recombinant proteins. From this, it was found that protein yields were greater for Autographa californica nuclear polyhedrosis virus (AcNPV) expression vectors using the basic protein promoter to drive production of JHE than for the p10 protein or polyhedrin promoters. This has significant implications for current baculovirus expression methodologies. The effect of multiplicity of infection on protein yield was found to be insignificant between 0.1 and 10 for expression under the basic protein promoter. Yields of JHE were about 40% higher from the cell line Tn5B1-4 ('High Five') relative to the Sf21 cell line under optimized conditions for each cell line, with maximum yields obtained at 2-3 days, and 3-5 days post-infection for the two cell lines respectively when cultured in ExCell 401 medium. The presence of fetal calf serum in the cell culture medium enhanced protein yields from both cell lines. These studies demonstrate the use of JHE as a reporter enzyme for optimizing high yields of protein from the baculovirus expression system. JHE also has a potential application as a reporter enzyme in other eukaryotic systems. The advantages and use of JHE over other reporter enzyme are discussed.
Bonning
B C
BC
Department of Entomology, University of California, Davis 95616, USA.
Hammock
B D
BD
D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D003470:Culture Media; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D017344:Genes, Insect; D017930:Genes, Reporter; D005814:Genes, Viral; D005822:Genetic Vectors; D007301:Insect Hormones / Q000235:genetics*; D009036:Moths / Q000235:genetics; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011401:Promoter Regions, Genetic; D018411:Spodoptera; D014773:Virology / Q000379:methods
D003470:Culture Media; D007301:Insect Hormones; D002265:Carboxylic Ester Hydrolases; C038003:juvenile hormone esterase