Construction of the mutated NHE3 proton exchanger and his function

Brauneger, Dora (2015) Construction of the mutated NHE3 proton exchanger and his function. Diploma thesis, Faculty of Science > Department of Biology.

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Abstract

The subject of this thesis was the construction of a mutated transmembrane isoform 3 Na+/H+ protein exchanger (NHE3). 24 amino acids deleted (Leu 586 – Arg609) are crucial for NHE3 protein regulation. The role of this exchanger is maintaining the homeostasis of cellular pH, by certain stimulators or inhibitors. The deletion has been accomplished by standard polymerase chain reaction method (PCR) using fragments of NHE3 sequence with flanking ends. The template for the reaction was pcDNA 3.1(+) plasmid with an inserted HA-NHE3 cDNA sequence (pcDNA/HA-NHE3). The efficacy of the deletion was checked by comparing the mutated fragment AD with control, unmutated fragment. After that, both pcDNA/HA-NHE3 plasmid and fragment AD were treated with PmaCI restriction enzyme having two restriction sites within the NHE3 sequence. Then, using the products of the reaction and treating them with ligase, a plasmid with a mutated NHE3 sequence was constructed, pcDNA/dHA-NHE3. Escherichia coli, DH5μ were transformed with ligation product and pcDNA/dHA-NHE3 plasmid was isolated from transformed colonies. The efficacy of the ligation and the proper orientation of the insert were examend by restriction analysis. The efficacy of the mutation was shown by PCR reaction using an anti-sense primer annealing to the deleted amino acid sequence. The final confirmation of the deletion was the sequencing of the isolated plasmid. A fibroblast PS120 cell line was transformed by pcDNA/dHA-NHE3 plasmid and the functionality of the NHE3 protein was shown by acid loading. The mutated protein was functional, leading to the conclusion that the protein contains several regulation sequences, separate or together.

Item Type: Thesis (Diploma thesis)
Keywords: NHE3, cloning
Supervisor: Žižak, Mirza
Co-supervisor: Bauer, Nataša
Date: 2015
Number of Pages: 71
Subjects: NATURAL SCIENCES > Biology
Divisions: Faculty of Science > Department of Biology
Depositing User: Grozdana Sirotic
Date Deposited: 16 Apr 2015 09:07
Last Modified: 16 Apr 2015 09:07
URI: http://digre.pmf.unizg.hr/id/eprint/3812

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