RESUMO
BACKGROUND: Recombinant protein purification is a crucial step for biochemistry and structural biology fields. Rapid robust purification methods utilize various peptide or protein tags fused to the target protein for affinity purification using corresponding matrices and to enhance solubility. However, affinity/solubility-tags often need to be removed in order to conduct functional and structural studies, adding complexities to purification protocols. RESULTS: In this work, the Vibrio cholerae MARTX toxin Cysteine Protease Domain (CPD) was inserted in a ligation-independent cloning (LIC) vector to create a C-terminal 6xHis-tagged inducible autoprocessing enzyme tag, called "the CPD-tag". The pCPD and alternative pCPD/ccdB cloning vectors allow for easy insertion of DNA and expression of the target protein fused to the CPD-tag, which is removed at the end of the purification step by addition of the inexpensive small molecule inositol hexakisphosphate to induce CPD autoprocessing. This process is demonstrated using a small bacterial membrane localization domain and for high yield purification of the eukaryotic small GTPase KRas. Subsequently, pCPD was tested with 40 proteins or sub-domains selected from a high throughput crystallization pipeline. CONCLUSION: pCPD vectors are easily used LIC compatible vectors for expression of recombinant proteins with a C-terminal CPD/6xHis-tag. Although intended only as a strategy for rapid tag removal, this pilot study revealed the CPD-tag may also increase expression and solubility of some recombinant proteins.
Assuntos
Clonagem Molecular/métodos , Cisteína Proteases/genética , Vetores Genéticos/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Vibrio cholerae/genética , Cisteína Proteases/isolamento & purificação , Histidina/genética , Proteínas Recombinantes de Fusão/isolamento & purificaçãoRESUMO
The Vibrio choleraeâ MARTXVc toxin delivers three effector domains to eukaryotic cells. To study toxin delivery and function of individual domains, the rtxA gene was modified to encode toxin with an in-frame beta-lactamase (Bla) fusion. The hybrid RtxA::Bla toxin was Type I secreted from bacteria; and then Bla was translocated into eukaryotic cells and delivered by autoprocessing, demonstrating that the MARTXVc toxin is capable of heterologous protein transfer. Strains that produce hybrid RtxA::Bla toxins that carry one effector domain in addition to Bla were found to more efficiently translocate Bla. In cell biological assays, the actin cross-linking domain (ACD) and Rho-inactivation domain (RID) are found to cross-link actin and inactivate RhoA, respectively, when other effector domains are absent, with toxin autoprocessing required for high efficiency. The previously unstudied alpha-beta hydrolase domain (ABH) is shown here to activate CDC42, although the effect is ameliorated when RID is also present. Despite all effector domains acting on cytoskeleton assembly, the ACD was sufficient to rapidly inhibit macrophage phagocytosis. Both the ACD and RID independently disrupted polarized epithelial tight junction integrity. The sufficiency of ACD but strong selection for retention of RID and ABH suggests these two domains may primarily function by modulating cell signaling.
Assuntos
Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Citoesqueleto/metabolismo , Vibrio cholerae/metabolismo , beta-Lactamases/metabolismo , Actinas/metabolismo , Resistência a Ampicilina , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Linhagem Celular , Linhagem Celular Tumoral , Polaridade Celular , Clonagem Molecular , Células HeLa , Humanos , Microtúbulos/metabolismo , Fagocitose , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/genética , beta-Lactamases/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Activation of inflammasomes is an important aspect of innate immune responses to bacterial infection. Recent studies have linked Vibrio cholerae secreted toxins to inflammasome activation by using murine macrophages. To increase relevance to human infection, studies of inflammasome-dependent cytokine secretion were conducted with the human THP-1 monocytic cell line and corroborated in primary human peripheral blood mononuclear cells (PBMCs). Both El Tor and classical strains of V. cholerae activated ASC (apoptosis-associated speck-like protein-containing a CARD domain)-dependent release of interleukin-1ß (IL-1ß) when cultured with human THP-1 cells, but the pattern of induction was distinct, depending on the repertoire of toxins the strains produced. El Tor biotype strains induced release of IL-1ß dependent on NOD-like receptor family pyrin domain-containing 3 (NLRP3) and ASC due to the secreted pore-forming toxin hemolysin. Unlike in studies with mouse macrophages, the MARTX toxin did not contribute to IL-1ß release from human monocytic cells. Classical biotype strains, which do not produce either hemolysin or the MARTX toxin, activated low-level IL-1ß release that was induced by cholera toxin (CT) and dependent on ASC but independent of NLRP3 and pyroptosis. El Tor strains likewise showed increased IL-1ß production dependent on CT when the hemolysin gene was deleted. In contrast to studies with murine macrophages, this phenotype was dependent on a catalytically active CT A subunit capable of inducing production of cyclic AMP and not on the B subunit. These studies demonstrate that the induction of the inflammasome in human THP-1 monocytes and in PBMCs by V. cholerae varies with the biotype and is mediated by both NLRP3-dependent and -independent pathways.