RESUMO
Broad-spectrum ß-lactam antibiotics such as penicillin are routinely used against both Gram-negative and Gram-positive bacteria. However, bacteria that produce ß-lactamase have developed resistance against these antibiotics by cleaving the ß-lactam ring and rendering the antibiotic inactive. To combat this effect, 1,4,7- Triazacyclononane (TACN), a cyclic organic compound derived from cyclononanes has been shown to preserve the activity of ß-lactam antibiotics by inhibiting ß-lactamase. However, its cytotoxic effects require elucidation. Given that the cytotoxic target for many therapeutics is the kidney, this study investigated the effects of TACN on human embryonic kidney cells (Hek293) cells. Hek293 cells were treated with TACN (0-500 µM) for 24 h and the cytotoxicity was assessed (MTT and LDH assay). Apoptosis was luminometrically detected by measuring phosphatidylserine externalisation and caspase activity and fluorescently detecting necrosis. DNA fragmentation was visualised using fluorescent microscopy. Expression of the apoptosis-related protein were determined by western blot. The results generated indicate that TACN does not initiate necrosis as LDH was decreased. Likewise, decreased apoptosis was supported by the decreased phosphatidylserine, caspases, Bax, cleaved PARP, IAP and NF-kB. However, increased DNA fragmentation was associated with increased p53. Therefore, effects of TACN at the nucleus, produced a p53 response to initiate DNA repair and did not culminate in cell death. The findings show that TACN is not cytotoxic to Hek293 cells via the apoptotic route. Since TACN did not induce cell death, its potential as a metallo-ß-lactamase inhibitor (MBLI) may be exploited to counteract the effect of MBL-producing bacteria. Restoring ß-lactam activity will curb the global menace of antibiotic resistance.
RESUMO
These investigations were designed to improve capture efficiency and selectivity in the immobilized metal ion affinity chromatographic (IMAC) purification of tagged recombinant proteins expressed in Escherichia coli cells, utilizing an alternative and novel class of immobilized metal binding ligands. The impact of loading conditions and lysate composition on the IMAC purification of NT1A- or His6 -tagged green fluorescent protein (GFP), using the ligands 1,4,7-triazacyclononane (tacn) and bis(1,4,7-triazacyclononyl)propane (dtnp), charged with Cu(2+) ions, has thus been explored. These findings were compared to the performance of a commercial adsorbent, IMAC Sepharose™ 6 FF, similarly charged with Cu(2+) ions. With the same loading, wash and elution protocols, the tacn- and dtnp-derived adsorbents showed higher selectivity in terms of removal of E. coli host cell proteins than the commercial adsorbent, while low molecular weight components in the crude lysate had a higher impact on the binding capacities of tacn- and dtnp-derived adsorbents. This effect of lysate composition could be reduced through osmotic shock treatment of the E. coli cells prior to lysis. Additionally, the protein-binding capacities of the tacn-based resins were enhanced by increasing their ligand densities. Because both the tacn- and the dtnp-derived IMAC adsorbents exhibit very high metal ion stability constants, under the chromatographic conditions examined, they could be used several times without re-charging with Cu(2+) ions. The results of these studies thus expand the general application scope of tacn-based IMAC resins for use in the capture and purification of tagged recombinant proteins.
Assuntos
Compostos Aza/química , Cromatografia de Afinidade/métodos , Proteínas de Fluorescência Verde/química , Piperidinas/química , Proteínas Recombinantes/isolamento & purificação , Compostos Aza/metabolismo , Quelantes/química , Quelantes/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/isolamento & purificação , Histidina/química , Ferro/química , Piperidinas/metabolismo , Ligação Proteica , Proteínas Recombinantes/químicaRESUMO
In this study, integration of three discrete process aspects of the IMAC purification of Escherichia coli expressed recombinant proteins has been investigated. To this end, novel N-terminally tagged human growth hormone variants (tagged-vhGHs) have been expressed in E. coli by tank fermentation and captured directly from the cell lysate by a new IMAC approach. The chelating ligands used were 1,4,7-triaza-cyclononane (tacn) and bis(1,4,7-triazacyclononyl)-propane (dtnp) with copper(II) as the immobilised metal ion. The N-terminal tags were specifically selected for their potential to bind to these immobilised complexes and also for their ease of removal from the tagged protein by the dipeptidyl peptidase, DAP-1. Low levels of detergents in the binding buffer did not dramatically affect the purification, but increased concentrations of NaCl in the loading buffer improved the binding performance. The same IMAC systems, operated in the 'negative' adsorption chromatographic mode, could be used to obtain the purified mature human growth hormone variant, as assessed by MALDI-TOF and N-terminal sequencing studies, following removal of the affinity tag by the dipeptidyl peptidase 1. Western immunoblot analysis of the eluted fractions of both the tagged and de-tagged vhGH demonstrated significant clearance of E. coli host cell proteins (HCPs). Further, these IMAC resins can be used multiple times without the need for metal ion re-charging between runs. This study thus documents an integrated approach for the purification of specifically tagged recombinant proteins expressed in genetically modified E. coli.