Recombinant expression and biochemical characterisation of two alanyl aminopeptidases of Trypanosoma congolense
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- 作者：Davita Pillay ; Alain F.V. Boulang茅 ; Virginie Coustou ; Th茅o Baltz ; Theresa H.T. Coetzer
- 关键词：AAT ; African animal trypanosomosis ; AEBSF ; 4-(2-aminoethyl)benzenesulfonyl fluoride ; AMT ; acetate-MES-Tris ; AMC ; aminomethylcoumarin ; AP ; amino peptidase ; BSF ; bloodstream form ; DAPI ; 4&prime ; 6-diamidino-2-phenylindole ; DMSO ; dimethyl sulfoxide ; DTT ; dithiothreitol ; E-64 ; L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane ; LC&ndash ; MS/MS ; liquid chromatography coupled to tandem mass spectrometry ; MBS ; m-maleimidobenzoyl-N-hydroxysuccinimide ester ; PBS ; phosphate buffered saline ; pH 7.2 ; PSG
- 刊名：Experimental Parasitology
- 出版年：December, 2013
- 年：2013
- 卷：135
- 期：4
- 页码：675-684
- 全文大小：1527 K
文摘
Trypanosoma congolense is a haemoprotozoan parasite that causes African animal trypanosomosis, a wasting disease of cattle and small ruminants. Current control methods are unsatisfactory and no conventional vaccine exists due to antigenic variation. An anti-disease vaccine approach to control T. congolense has been proposed requiring the identification of parasitic factors that cause disease. Immunoprecipitation of T. congolense antigens using sera from infected trypanotolerant cattle allowed the identification of several immunogenic antigens including two M1 type aminopeptidases (APs). The two APs were cloned and expressed in Escherichia coli. As the APs were expressed as insoluble inclusion bodies it was necessary to develop a method for solubilisation and subsequent refolding to restore conformation and activity. The refolded APs both showed a distinct substrate preference for H-Ala-AMC, an optimum pH of 8.0, puromycin-sensitivity, inhibition by bestatin and amastatin, and cytoplasmic localisation. The two APs are expressed in procyclic metacyclic and bloodstream form parasites. Down-regulation of both APs by RNAi resulted in a slightly reduced growth rate in procyclic parasites in vitro.