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Title: A study of actinidin expression in yeast
Author: Yuwono, Triwibowo
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 1991
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Four different actinidin gene constructions have been created, each consisting of different functional parts of the actinidin gene: (1) the mature actinidin-coding DNA, (2) the amino-terminal extension, but without the secretion signal, plus the mature actinidin-coding DNA, (3) the mature actinidin plus the carboxy-terminal extension-coding DNA, and (4) the full-length precursor actinidin-coding DNA. The first three constructions were fused to the yeast MFa1 promoter and secretion leader sequence, while the fourth was coupled to the CYC1-GAL UAS promoter. Upon expression in yeast, no protein product was detected in the culture supernatant. Analysis of intracellular proteins showed that actinidin protein was detected only from the actinidin gene constructions which have the carboxy-terminal extensions, suggesting that the carboxy-terminal extension is required for the stability of the protein. Comparison of the actinidin proteins produced in protease-proficient and protease-deficient strains suggests that the processing of the protein requires the activity of vacuolar protease(s) and indicates that the actinidin was translocated into the yeast vacuole. Examination of the amino acid sequence suggested that actinidin possesses potential vacuolar and peroxisomal targeting signals. Since the actinidin precursor was glycosylated it must have entered the secretory pathway before being translocated into a specific cellular compartment. The HSP26 gene promoter has been shown to be induced by heat-shock and upon entry into stationary phase, thus it is potentially useful for heterologous gene expression in yeast.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available