Browsing by Author "Roszik, Lilla"
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- The BiP Chaperone from the Endoplasmic Reticulum of Zebrafish (Daniorerio) and Golden Hamster (Mesocricetus auratus)Publication . Roszik, Lilla; Melo, Eduardo PinhoThe Hsp70 family of molecular chaperones plays a crucial role in maintaining cellular proteostasis by promoting protein folding, transport, and degradation. One of its component proteins, the binding immunoglobulin protein (BiP), is an essential endoplasmic reticulum chaperone required for proper protein folding and stress response. The primary objective of this work was to clone the open reading frame of the BiP gene from zebrafish (Danio rerio), a model organism that is frequently used to study the molecular and genetic causes of human disorders. More than 70% of the genes between humans and zebrafish are similar, including many orthologs connected to neurodegenerative illnesses like Alzheimer's. For the sake of further functional investigations, the open reading frame of the zebrafish BiP gene was amplified and inserted into a bacterial expression vector. From zebrafish, the total RNA of caudal cell lines was extracted and reverse transcribed to create complementary DNA. The target DNA sequence was amplified by polymerase chain reaction (PCR) using specific primers for BiP. The golden hamster BiP ORF was removed from the backbone pQE10, and the amplified product was cloned into the vector. After transforming the recombinant plasmid into Escherichia coli DH5α for propagation and isolation, the insertion of the open reading frame of zebrafish BiP was sent to sequencing. Unfortunately, E coli recombines the zebrafish BiP ORF, contrary to the golden hamster ORF previously cloned into the same vector. Different E coli strains deficient in DNA recombination should be tested soon. This work aimed to establish the foundation for further investigations into the activity and biochemical characteristics of zebrafish BiP and its interactions with co-chaperones and client proteins in comparison with the mammalian BiP from golden hamster. At the longer term, we want to understand how the chaperone machinery of the endoplasmic reticulum has evolved in resolving protein aggregates associated with neurodegenerative diseases.