Expression and Purification of Recombinant VMO1 Protein
Hardie, S. E. (2015). Expression and Purification of Recombinant VMO1 Protein (Thesis, Master of Science (Research) (MSc(Research))). University of Waikato, Hamilton, New Zealand. Retrieved from http://hdl.handle.net/10289/9609
Permanent Research Commons link: http://hdl.handle.net/10289/9609
Hearing loss is a common sensory deficit that affects more than 275 million people worldwide. Identifying and understanding the genetic causes which underlie hearing loss can lead to improved diagnostic and treatment strategies. The biological function of Vitelline Membrane Outer Layer 1 homolog (chicken) (VMO1) is of particular interest as the mRNA transcript was previously found to be uniquely expressed and highly abundant in the mouse inner ear. The aim of this research was to design, clone, express and purify a recombinant VMO1 protein containing a His Tag using the VMO1 gene amplified from human cell lines. The VMO1 gene was cloned into the pET28b(+) expression system and bacterial cells were manipulated to express the recombinant VMO1 protein through IPTG induction. The resulting recombinant protein was then purified using a nickel affinity gel. Finally, a commercial VMO1 antibody was tested and validated using the purified recombinant VMO1 protein using western blot analysis. Bioinformatics analysis was undertaken to develop primers to amplify cDNA of two isoforms of the VMO1 gene in human cell lines, isoform 1 and isoform 3. Both isoforms of the VMO1 gene were found to be expressed in two commercial in vitro human cancer cell lines; monocytic leukaemia and breast cancer. Following amplification, the isoforms were sub cloned into T-tailed vectors, with isoform 1 being successfully ligated into the pET28b(+) expression vector. Sequencing data confirmed the amplified cloned PCR inserts were VMO1 transcripts, and the predicted translated sequence was in-frame with the His Tag, resulting in a recombinant protein that was 20 kDa in size. Expression of the recombinant VMO1 protein was attempted using three bacterial expression strains with successful expression of a 20 kDa protein occurring in the Rosetta™ (DE3) pLysS strain. The recombinant VMO1 protein formed inclusion bodies within bacterial cells following over expression. Western blot analysis of non purified protein samples showed that the 6X His antibody recognised a 20 kDa recombinant protein as expected. However, the commercially available VMO1 antibody detected two bands approximately 20 and 35 kDa in size. In the future, it is recommended that optimisation of recombinant protein folding methodology, large scale protein induction and purification of the VMO1 protein be completed. In addition, it is recommended to validate the VMO1 antibody by testing in vitro human cancer cell lines for the expression of VMO1 protein. Once the specificity of the VMO1 antibody has been demonstrated by western blot analysis, functional assays could be performed to determine the biological function of VMO1. This may provide further insight into the function of VMO1 in the mammalian inner ear and the role it may play in hearing.
University of Waikato
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