The expression and purification of Rukh61 and Mb
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The protein Rukh61 was expressed in the fusion vector pGEX-4T-1 and the recombinant organism grown in Luria broth and minimal media. Ruk is expressed early in rat development, in a variety of tissues. Ruk is of interest because of its interaction with proteins in the signal transduction pathway. It is known to inhibit a PI3-kinase pathway which involves Akt/PKB. The mode of action of this protein is of interest because it may lead to new anti-cancer drugs. The protein was purified using affinity chromatography. Thrombin protease was used to cleave the GST tag from the target protein and affinity chromatography used to remove the GST tag from the solution containing the target protein. Concentration of the protein has been achieved through the use of column chromatography, the freeze drying of samples, and by ammonium sulphate precipitation. Through the course of the work, the protocol for the purification of GST-Rukh61 has been optimised, as has the protocol for the cleavage of the GST tag and the concentration of the protein. The protein GST-Mb has also been expressed in small quantities. Mb is of interest because it is found in mice in neuronal tissue only and it is only found during the later stages of development. Mb responds to nerve growth factors thus its mode of action, investigated through structural studies, is of interest. It has been transformed into a different Escherichia coli (E. coli) strain, from that in which it was obtained, to facilitate expression. The Mb can also be cleaved from the GST using thrombin protease. Work on the optimisation of the protocols for Mb is ongoing.
Thesis, MPhil Master of Philosophy
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