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Cloning, production and purification of proteins that interact with Apoptin

Cancer is a group of pathologies of clonal origin that appears due to an accumulation of mutations in an organism. It is characterized by an abnormal growth of cells that tend to proliferate in an uncontrolled way leading to the formation of a mass or tissue accumulation commonly known as tumor. Cancer is one of the main causes of mortality worldwide. Current cancer treatments can be invasive and sometimes fail at being efficient. For this reason, research is currently focused on targeted therapies. For instance, treatment with Apoptin, which specifically kills cancer cells leaving non-cancer cells unharmed. Apoptin is a protein coded by the genome of the chicken anemia virus that specifically kills tumor cells. This protein has a strong tendency to aggregate and is a member of the intrinsically disordered proteins, a group of proteins that do not have a defined structure and can interact with several proteins by conformational changes. The aim of this Final Grade Project forms part of a wider project that consists in studying the structural changes that Apoptin suffers when interacting with proteins related to cellular signaling in order to find out more about the molecular basis of the mechanism of cytotoxicity of this protein. In this project I have cloned two proteins that are known to interact with Apoptin. These proteins are Breast cancer associated gene 3 and Peptidyl-prolyl isomerase-like 3 which have been fused to Glutathione S-transferase. For this reason, each gene has been designed choosing the optimal codon usage and introducing the appropriate restriction sites with the aim of cloning them in pGEX-4T-2 vector. This vector contains the DNA sequence that codes for Trombine, which can be used for the removal of Glutathione S-transferase from the recombinant protein. Once the clonation has been finished, the recombinant proteins have been produced in Escherichia coli strain XL1Blue and a variant of Apoptin has been purified. This variant is called H6-ApoptinΔProΔLeu and its residues 1 to 43 have been eliminated in order to reduce its tendency to aggregate without affecting its cytotoxic effect on cancer cells. Once purified, pull-down assays have been performed with the aim of investigating the in vitro interaction of these proteins with Apoptin

Manager: Benito i Mundet, Antoni
Other contributions: Universitat de Girona. Facultat de Ciències
Author: Vivet Noguer, Raquel
Date: 2016 June
Abstract: Cancer is a group of pathologies of clonal origin that appears due to an accumulation of mutations in an organism. It is characterized by an abnormal growth of cells that tend to proliferate in an uncontrolled way leading to the formation of a mass or tissue accumulation commonly known as tumor. Cancer is one of the main causes of mortality worldwide. Current cancer treatments can be invasive and sometimes fail at being efficient. For this reason, research is currently focused on targeted therapies. For instance, treatment with Apoptin, which specifically kills cancer cells leaving non-cancer cells unharmed. Apoptin is a protein coded by the genome of the chicken anemia virus that specifically kills tumor cells. This protein has a strong tendency to aggregate and is a member of the intrinsically disordered proteins, a group of proteins that do not have a defined structure and can interact with several proteins by conformational changes. The aim of this Final Grade Project forms part of a wider project that consists in studying the structural changes that Apoptin suffers when interacting with proteins related to cellular signaling in order to find out more about the molecular basis of the mechanism of cytotoxicity of this protein. In this project I have cloned two proteins that are known to interact with Apoptin. These proteins are Breast cancer associated gene 3 and Peptidyl-prolyl isomerase-like 3 which have been fused to Glutathione S-transferase. For this reason, each gene has been designed choosing the optimal codon usage and introducing the appropriate restriction sites with the aim of cloning them in pGEX-4T-2 vector. This vector contains the DNA sequence that codes for Trombine, which can be used for the removal of Glutathione S-transferase from the recombinant protein. Once the clonation has been finished, the recombinant proteins have been produced in Escherichia coli strain XL1Blue and a variant of Apoptin has been purified. This variant is called H6-ApoptinΔProΔLeu and its residues 1 to 43 have been eliminated in order to reduce its tendency to aggregate without affecting its cytotoxic effect on cancer cells. Once purified, pull-down assays have been performed with the aim of investigating the in vitro interaction of these proteins with Apoptin
Format: application/pdf
Document access: http://hdl.handle.net/10256/12974
Language: eng
Collection: Biotecnologia (TFG)
Rights: Attribution-NonCommercial-NoDerivs 3.0 Spain
Rights URI: http://creativecommons.org/licenses/by-nc-nd/3.0/es/
Subject: Apoptosi
Enginyeria de proteïnes
Medicaments anticancerosos
Proteïnes supressores de tumors
Càncer
Cancer
Tumor suppressor proteins
Anticancer drugs
Protein engineering
Apoptosis
Title: Cloning, production and purification of proteins that interact with Apoptin
Type: info:eu-repo/semantics/bachelorThesis
Repository: DUGiDocs

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