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Cloning & Expression of Centrosome Kinase, Polo- Like Kinase 4 (PLK4) by Umama Shahid

By: Contributor(s): Material type: TextTextPublication details: IIT Jodhpur Department of Bioscience and Bioengineering 2019Description: xviii,54p. HBSubject(s): DDC classification:
  • 571.65 Sh13C
Summary: Centrosome is a well-known microtubule organizing center in various animal cells. It has been associated with plethora of cellular functions like cell division, cell migration, cell polarity and ciliogenesis. The number of centrosomes per cell is highly regulated in a cell cycle dependent manner. Polo-Like Kinase 4 (PLK4) is a master regulator involved in centrosome duplication during the cell cycle. Centrosome dysfunction has been associated with several human diseases like primary microcephaly and cancers, thereby emphasizing the need to get a clear understanding of the molecular pathways regulating centriole number and functioning. The objective of this thesis was to clone and express PLK4 using a bacterial expression system. Classical cut and paste cloning method has been used in order to introduce PLK4 cDNA in pET Duet1 vector, a bacterial expression vector. The recombinant plasmid was used to express PLK4 in bacteria, E. coli BL21 cells. The recombinant purified PLK4 protein is useful to investigate the details and dynamics of its interaction with other centrosome proteins.Keywords: Centrosome, Polo-Like Kinase 4 , cloning, pET Duet1 vector, Protein expression
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Thesis Thesis S. R. Ranganathan Learning Hub Course Reserve Reference 571.65 Sh13C (Browse shelf(Opens below)) Not for loan TM00149
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Centrosome is a well-known microtubule organizing center in various animal cells. It has been associated with plethora of cellular functions like cell division, cell migration, cell polarity and ciliogenesis. The number of centrosomes per cell is highly regulated in a cell cycle dependent manner. Polo-Like Kinase 4 (PLK4) is a master regulator involved in centrosome duplication during the cell cycle. Centrosome dysfunction has been associated with several human diseases like primary microcephaly and cancers, thereby emphasizing the need to get a clear understanding of the molecular pathways regulating centriole number and functioning. The objective of this thesis was to clone and express PLK4 using a bacterial expression system. Classical cut and paste cloning method has been used in order to introduce PLK4 cDNA in pET Duet1 vector, a bacterial expression vector. The recombinant plasmid was used to express PLK4 in bacteria, E. coli BL21 cells. The recombinant purified PLK4 protein is useful to investigate the details and dynamics of its interaction with other centrosome proteins.Keywords: Centrosome, Polo-Like Kinase 4 , cloning, pET Duet1 vector, Protein expression

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