Application of Adenovirus Expression System for the Production of Recombinant Cholinesterases
Abstract
Previous studies in rodents and nonhuman primates demonstrated that pretreatment of animals with cholinesterases (ChEs) could provide significant protection against behavioral and lethal effects of nerve agent intoxication. Currently, human serum butyrylcholinesterase (BChE) is under development as medical countermeasure against organophosphate chemical warfare agent toxicity. In this study, we report the expression of human BChE and bovine acetylcholinesterase (AChE) using the newly designed adenovirus expression system. The shuttle vector employed in this expression system contains cytomegalovirus-5 (CMV-5) promoter that was 5 to 10 times more efficient than the original CMV-1 promoter in inducing transgene expression in human embryonic epithelial cell line, namely 293 A. Both BChE and AChE were fully expressed as tetrameric forms by the simultaneous expression of proline-rich attachment domain in 293 A cells. By optimizing the culture conditions, 12-15 U/ml of AChE and 1.5-2.0 U/ml of BChE were produced with this system. The expressed rHu BChE was purified to homogeneity by a two-step procedure involving procainamide affinity column and nickel affinity column. The physico-chemical properties of BChE were found to be similar to those of the native protein. In addition, purified recombinant BChE showed catalytic properties similar to those of the native protein. These studies suggest that it is possible to produce milligram to gram quantities of homogeneous preparations of recombinant tetrameric AChE and BChE in tissue culture using the adenovirus expression system.
Document Details
- Document Type
- Technical Report
- Publication Date
- Oct 01, 2005
- Accession Number
- ADA449747
Entities
People
- Akrati Saxena
- B. P. Doctor
- C. Chambers
- J. M. Watt
- K. Parikh
- N. Chilukuri
- P. Tipparaju
- R. Naik
Organizations
- Walter Reed Army Institute of Research