Expression of a bee venom phospholipase A2 from Apis cerana cerana in the baculovirus-insect cell.
Bee venom phospholipase A(2) (BvPLA(2)) is a lipolytic enzyme that catalyzes the hydrolysis of the sn-2 acyl bond of glycerophospholipids to liberate free fatty acids and lysophospholipids. In this work, a new BvPLA(2) (AccPLA(2)) gene from the Chinese honeybee (Apis cerana cerana) venom glands was inserted into bacmid to construct a recombinant transfer vector. Tn-5B-4 (Tn) cells were transfected with the recombinant bacmid DNA for expression. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed a double band with molecular weights of 16 and 18 kDa. Products of hexahistidine AccPLA(2) fusion protein accumulated up to 5.32% of the total cellular proteins. The AccPLA(2) fusion protein was cross reactive with the anti-AmPLA(2) (BvPLA(2) of the European honeybee, Apis mellifera) polyclonal serum. The reaction resulted in a double glycosylation band, which agrees with the band generated by the native AmPLA(2) in Western blot analysis. The PLA(2) activity of the total extracted cellular protein in the hydrolyzing egg yolk is about 3.16 micromol/(min.mg). In summary, the recombinant AccPLA(2) protein, a native BvPLA(2)-like structure with corresponding biological activities, can be glycosylated in Tn cells. These findings provided fundamental knowledge for potential genetic engineering to produce AccPLA(2) in the pharmaceutical industry.
Shen
Li-Rong
LR
Department of Food Science and Nutrition, Zhejiang University, Hangzhou 310029, China. shenlirong@zju.edu.cn
Ding
Mei-Hui
MH
Zhang
Li-Wen
LW
Zhang
Wei-Guang
WG
Liu
Liang
L
Li
Duo
D
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001514:Bee Venoms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001516:Bees / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D004795:Enzyme Stability; D005822:Genetic Vectors / Q000235:genetics; D007313:Insecta; D054467:Phospholipases A2 / Q000737:chemistry* / Q000502:physiology*; D015202:Protein Engineering / Q000379:methods*; D014162:Transfection / Q000379:methods
D001514:Bee Venoms; D054467:Phospholipases A2