The human proton channel Hv1 (hHv1) is a voltage-gated ion channel, and it is known for its involvement with several different physio pathological processes, such as the emergence of tissue acidosis as a consequence of ischemic events, the homeostasis of tumor microenvironmental acidity and metastasis. Overexpression of hHv1 has been identified in both breast and colorectal cancer, the most common gender specific neoplastic pathology in the population of industrialised economies. Despite the clinical and epidemiological relevance of these pathologies, and the acknowledged and effective pharmacology directed against other voltage-gated ion channels, the pharmacology on hHv1 remains, notwithstanding recent developments, substantially limited. This is partially due to the lack of structural information on the human channel, caused by the absence of a crystallographic model of hHv1. This work describes the purification process for the human voltage gated proton channel hHv1, through two different approaches in affinity chromatography. We compared the conventional purification approach, Immobilised Metal Affinity Chromatography (IMAC), characterised by the interaction between an histidine tag fused to our protein of interest, and metallic ions immobilised on resin, with an unconventional approach in affinity chromatography, based on a Sepharose resin loaded with Anti-GFP nanobodies, camelid antibodies directed against the GFP fusion protein. The results show that the use of camelid antibodies is the most effective strategy to purify the hHv1 channel. This can probably be ascribed to a the higher affinity of the nanobodies for the GFP epitope fused with hHv1, that predisposes this chromatographic setup to a fewer number of non-specific or undesired interactions. The protocol we developed is a single-step purification method that significantly reduce the time necessary to obtain an homogeneous protein sample. To date electrophysiological studies and ligand binding assays are necessary to deepen our knowledge of this ion channel: it is believed that a robust and effective purification process for this protein may constitute a step towards the achievement of that objective.
Il canale protonico umano Hv1 (hHv1) è un canale voltaggio-dipendente implicato in numerosi processi fisiopatologici, come nell’emergenza dell’acidosi tissutale nel danno ischemico o nel mantenimento del microambiente tumorale e nella metastasi. La sovraespressione di hHv1 è stata identificata sia nel tumore al seno che in quello al colon retto, rispettivamente le patologie oncologiche più diffuse nella popolazione femminile e maschile appartenenti ad economie industrializzate. Nonostante la rilevanza clinica ed epidemiologica delle patologie coinvolte, e l’esistenza di una riconosciuta ed efficace farmacologia diretta nei confronti di canali ionici e proteine di membrana, la farmacologia riguardante hHv1 resta, malgrado i recenti sviluppi, fondamentalmente limitata. Questo è imputabile, almeno in parte alla mancanza di informazioni strutturali precise sul canale umano, dovuta all’assenza della risoluzione di un modello cristallografico per il canale protonico umano. Questo lavoro di tesi descrive il processo di purificazione del canale umano Hv1 attraverso due approcci in cromatografia di affinità. Abbiamo confrontanto per processo e risultati l’approccio convenzionale di Immobilised Metal Affinity Chromatography (IMAC), caratterizzato dall’interazione di un tag istidinico con ioni metallici immobilizzati su resina, con un approccio in cromatografia ad affinità non convenzionale, basato su resina di Sefarosio caricata con Nanobody Anti GFP , ovvero anticorpi camelidi diretti contro la proteina di fusione GFP. Dalle prove effettuate è emersa una maggiore efficacia della procedura che utilizza i nanobody anti GFP nella purificazione di hHv1, probabilmente ascrivibile ad una maggiore affinità degli anticorpi per l’epitopo di GFP fuso con hHv1, che predispone questo setup cromatografico ad un minor numero di interazioni aspecifiche o indesiderate. Questo approccio permette di arrivare ad un risultato purificativo soddisfacente in un minor numero di passaggi, riducendo in modo significativo i tempi necessari per produrre un campione omogeneo. Ad oggi, studi di elettrofisiologia e saggi di binding sono necessari per approfondire la conoscenza di questo canale ionico: si ritiene che la descrizione di un processo purificativo robusto ed efficace possa costituire un passo in questa direzione.
Strategie di Purificazione del canale protonico umano Hv1 (hHv1)
FONTANA, FRANCESCO
2020/2021
Abstract
The human proton channel Hv1 (hHv1) is a voltage-gated ion channel, and it is known for its involvement with several different physio pathological processes, such as the emergence of tissue acidosis as a consequence of ischemic events, the homeostasis of tumor microenvironmental acidity and metastasis. Overexpression of hHv1 has been identified in both breast and colorectal cancer, the most common gender specific neoplastic pathology in the population of industrialised economies. Despite the clinical and epidemiological relevance of these pathologies, and the acknowledged and effective pharmacology directed against other voltage-gated ion channels, the pharmacology on hHv1 remains, notwithstanding recent developments, substantially limited. This is partially due to the lack of structural information on the human channel, caused by the absence of a crystallographic model of hHv1. This work describes the purification process for the human voltage gated proton channel hHv1, through two different approaches in affinity chromatography. We compared the conventional purification approach, Immobilised Metal Affinity Chromatography (IMAC), characterised by the interaction between an histidine tag fused to our protein of interest, and metallic ions immobilised on resin, with an unconventional approach in affinity chromatography, based on a Sepharose resin loaded with Anti-GFP nanobodies, camelid antibodies directed against the GFP fusion protein. The results show that the use of camelid antibodies is the most effective strategy to purify the hHv1 channel. This can probably be ascribed to a the higher affinity of the nanobodies for the GFP epitope fused with hHv1, that predisposes this chromatographic setup to a fewer number of non-specific or undesired interactions. The protocol we developed is a single-step purification method that significantly reduce the time necessary to obtain an homogeneous protein sample. To date electrophysiological studies and ligand binding assays are necessary to deepen our knowledge of this ion channel: it is believed that a robust and effective purification process for this protein may constitute a step towards the achievement of that objective.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/14686