High-level expression of functional recombinant human butyrylcholinesterase in silkworm larvae by Bac-to-Bac system
Publication in refereed journal


引用次數
替代計量分析
.

其它資訊
摘要Butyrylcholinesterase (BChE: EC 3.1.1.8) serves as a natural scavenger for a variety of drugs, poisons, and organophosphorous compounds by hydrolyzing their ester bonds. Large scale production of recombinant human BChE (rhBChE) has been reported in transgenic goat. Here we demonstrate high-level expression of rhBChE with biological activity comparable to that of natural and recombinant enzymes, through the Bac-to-Bac baculovirus expression system in silkworm Bombyx mori larvae. We constructed the full-length hBChE cDNA into the plasmid pFastBac (TM). To monitor the level of expression, the cDNA coding for an orange fluorescent protein (OFP) was cloned downstream to the polyhedron (pH) promoter. Transfection was carried out by subcutaneous injection of 4-5th instar silkworm larvae. Approximately 4-7 days after infection, high-level expression of recombinant proteins was observed as indicated by the orange fluorescence of the larvae under blue light illumination. The hemolymph of the infected larvae was harvested, purified and assayed for BChE activity. The total units of BChE activity after purification were around 6.4 units per larvae. The K(m) and V(max) values of rhBChE were determined to be 17.7 mu M and 2194 U/l hemolymph, respectively. By SDS-PAGE and Western analysis, the size of silkworm rhBChE was estimated to be 85 kDa. The results indicate that the silkworm larva is a good alternative system to produce bioactive rhBChE. Further optimization and modifications will be necessary for large-scale production of rhBChE. This should provide a rapid, low-cost, and high yield rhBChE for therapeutic applications. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
著者Li S, Ip DTM, Lin HQ, Liu JM, Miao YG, Ke LJ, Wan DCC
會議名稱10th International Meeting on Cholinesterases
會議開始日20.09.2009
會議完結日25.09.2009
會議地點Sibenik
期刊名稱Chemico-Biological Interactions
出版年份2010
月份9
日期6
卷號187
期次1-3
出版社Elsevier
頁次101 - 105
國際標準期刊號0009-2797
電子國際標準期刊號1872-7786
語言英式英語
關鍵詞Bac-to-Bac system; Butyrylcholinesterase; Purification; Silkworm (Bombyx mori L.)
Web of Science 學科類別Biochemistry & Molecular Biology; BIOCHEMISTRY & MOLECULAR BIOLOGY; Pharmacology & Pharmacy; PHARMACOLOGY & PHARMACY; Toxicology; TOXICOLOGY

上次更新時間 2021-23-01 於 00:41