Heterologous Expression Of Avian Influenza A (H5n1) Neuraminidase In Kluyveromyces lactis AND Escherichia coli

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dc.contributor.authorTaufik, Noor Zafiah
dc.date.accessioned2017-01-04T06:31:53Z
dc.date.available2017-01-04T06:31:53Z
dc.date.issued2016-09
dc.description.abstractThe expression of H5N1 virus neuraminidase (NA) in simple microbial expression system utilizing Kluyveromyces lactis and Escherichia coli as the host was studied. For NA protein production in K. lactis, two types of NA DNA fragment composed of 1) full length sequence (1347 bp) and 2) head domain sequence (residue 63-449, 1161 bp) from A/Chicken/Malaysia/5858/2004 (H5N1) were amplified and cloned into pKLAC2 expression vector generating plasmid constructs named pKLAC2-NA and pKLAC2-NAHD. Both plasmid were then linearized and integrated into the LAC4 promoter region of K. lactis genome through homologous recombination. However, protein analysis carried out upon the induction of protein expression did not exhibit the presence of secreted recombinant NA (rNA) protein by any of the yeast transformants. Hence, the full length sequence of NA gene was also amplified and cloned into pET-32 Xa/LIC expression vector and subsequently used to transform E. coli Rosetta-gami 2(DE3). Protein analysis performed after the protein expression induction shows that ~ 62 kDa size rNA protein was expressed in the form of inclusion bodies (IBs). Next, to recover the bioactive rNA protein, the IBs were isolated, solubilized and refolded using pulsatile dilution method. The effects of pH, temperature, and chemicals additives were also investigated. At least 27% of the solubilized IBs were successfully refolded, and low amount of enzyme activity was recorded.en_US
dc.identifier.urihttp://hdl.handle.net/123456789/3308
dc.subjectH5N1 virus neuraminidase (NA) in simple microbial expression systemen_US
dc.subjectutilizing Kluyveromyces lactis and Escherichia colien_US
dc.titleHeterologous Expression Of Avian Influenza A (H5n1) Neuraminidase In Kluyveromyces lactis AND Escherichia colien_US
dc.typeThesisen_US
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