RESUMO
We present here three expression plasmids for Trypanosoma cruzi adapted to the Gateway® recombination cloning system. Two of these plasmids were designed to express trypanosomal proteins fused to a double tag for tandem affinity purification (TAPtag). The TAPtag and Gateway® cassette were introduced into an episomal (pTEX) and an integrative (pTREX) plasmid. Both plasmids were assayed by introducing green fluorescent protein (GFP) by recombination and the integrity of the double-tagged protein was determined by western blotting and immunofluorescence microscopy. The third Gateway adapted vector assayed was the inducible pTcINDEX. When tested with GFP, pTcINDEX-GW showed a good response to tetracycline, being less leaky than its precursor (pTcINDEX).
Assuntos
Expressão Gênica/genética , Vetores Genéticos/genética , Plasmídeos , Mapeamento por Restrição/métodos , Trypanosoma cruzi/genética , Western Blotting , Etiquetas de Sequências Expressas/metabolismo , Proteínas de Fluorescência Verde , Estágios do Ciclo de Vida/genética , Mutagênese Insercional , Tetraciclina/farmacologia , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Objective To clone and express Dengue virus nonstructural protein 4A (NS4A) gene and express in eukaryotic cells.Then,to isolate and purify and isolate cellular proteins interacted with NS4A.Methods With specific primers,NS4A gene fragment tagged with FLAG and HA (FLAG-NS4A-HA) was amplified by PCR and cloned into an expression vector,pSG5 vector.Recombinant plasmid was transfected into A549 cells by LipofectAMINETM2000.Transient expression of FLAG-NS4A-HA was detected by Western blot.The NS4A interacting proteins were isolated and purified by tandem affinity purification (TAP) system using HA and FLAG antibodies,and then assayed by silver stained SDS-PAGE.Results Dengue virus NS4A gene tagged with FLAG and HA was successfully constructed into pSG5 vector and expressed in A.549 cells.Silver stained SDS-PAGE showed that the expressed NS4A and two potential interacting proteins that interact with NS4A were isolated after TAP purification and SDS-PAGE.Conclusion Cellular proteins that potentially interacted with Dengue virus NS4A were successfully purified and isolated,which provided a basis for further research.
RESUMO
It's a multi-protein complex and not single protein that accomplish most of cellular activity.Therefore,to identify and analyze the composition of protein complex is necessary for studying the function of proteins.Tandem affinity purification technique(TAP) enables the effective purification of complex under physiological conditions without knowledge of the complex composition and function.Now,it has been adapted to analyze the composition of the protein complexes in yeast.Combined with mass spectrometry,TAP can identify the interacting proteins of target protein and offer great help for opening protein interacting network and function out.