title: Expression and Purification of Recombinant VMO1 Protein

authors: Hardie, Sarah Elyse

subjects: VMO1

abstract: 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.

text: Expression and Purification of Recombinant VMO1 Protein 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.

year: 2015

title: Aspects of protease production by Thermus strain Ok6 and other New Zealand isolates

authors: Jones, Colin W.

subjects: biology

abstract: Thirteen strains of Thermus isolated from New Zealand hot pools were screened for their ability to grow on minimal or near-minimal media and to exhibit extracellular protease activity. Environmental regulation of protease production by one such strain, Thermus Ok6, was investigated in detail. Protease activity was maximally derepressed during growth under conditions which lead to energy starvation (i.e. highly oxidized carbon substrate, oxygen limitation, slow growth rate and a high external pH), and was variably repressed by different nitrogen sources. These results suggest that the function of the enzyme is to scavenge carbon/energy and nitrogen. The protease was loosely attached to the cell under these growth conditions but could be released by growing the organism in high concentrations of trypticase peptone plus yeast extract, by exposing the culture to moderate shear forces or by washing cells in high ionic strength solutions of various inorganic salts, organic acids or amino acids. A simple and rapid procedure for the partial purification of the enzyme is described.

text: Aspects of protease production by Thermus strain Ok6 and other New Zealand isolates Thirteen strains of Thermus isolated from New Zealand hot pools were screened for their ability to grow on minimal or near-minimal media and to exhibit extracellular protease activity. Environmental regulation of protease production by one such strain, Thermus Ok6, was investigated in detail. Protease activity was maximally derepressed during growth under conditions which lead to energy starvation (i.e. highly oxidized carbon substrate, oxygen limitation, slow growth rate and a high external pH), and was variably repressed by different nitrogen sources. These results suggest that the function of the enzyme is to scavenge carbon/energy and nitrogen. The protease was loosely attached to the cell under these growth conditions but could be released by growing the organism in high concentrations of trypticase peptone plus yeast extract, by exposing the culture to moderate shear forces or by washing cells in high ionic strength solutions of various inorganic salts, organic acids or amino acids. A simple and rapid procedure for the partial purification of the enzyme is described.

year: 1988

title: The perspective of youth leadership through the insight of current Māori leaders

authors: Wairepo, Ngareta

abstract: Within Te Ao Māori, leadership qualities are central to Māori cultural norms and societal structures. In particular, a rich legacy of supporting intergenerational leadership exists, wherein following generations are endorsed into these leadership roles. Yet, little is documented in academic literature regarding first-hand experiences of the relationship between current Māori leader’s opinions, experiences, and perceptions for supporting the development of future and upcoming rangatahi into leadership roles. Therefore, this research project seeks to explore how leadership and the value of intergenerational indigenous wisdom can contribute to the supporting and flourishing of rangatahi into leadership roles and responsibilities in their communities to support intergenerational leadership development. 
A Kaupapa Māori approach was used for the framework to centre Indigenous ways of being and knowing. Eight semi-structured, conversational interviews were conducted with current Māori leaders to understand their perceptions, experiences, and opinions on leadership. These were then analysed using thematic analysis. Three major themes were identified; Mental strategies, Understanding the complexity of Māori leadership, and Intergenerational development. This research affirms the importance of leadership for Māori communities, and in particular, the role of intergenerational knowledge-sharing as key for supporting rangatahi to be well.

text: The perspective of youth leadership through the insight of current Māori leaders Within Te Ao Māori, leadership qualities are central to Māori cultural norms and societal structures. In particular, a rich legacy of supporting intergenerational leadership exists, wherein following generations are endorsed into these leadership roles. Yet, little is documented in academic literature regarding first-hand experiences of the relationship between current Māori leader’s opinions, experiences, and perceptions for supporting the development of future and upcoming rangatahi into leadership roles. Therefore, this research project seeks to explore how leadership and the value of intergenerational indigenous wisdom can contribute to the supporting and flourishing of rangatahi into leadership roles and responsibilities in their communities to support intergenerational leadership development. 
A Kaupapa Māori approach was used for the framework to centre Indigenous ways of being and knowing. Eight semi-structured, conversational interviews were conducted with current Māori leaders to understand their perceptions, experiences, and opinions on leadership. These were then analysed using thematic analysis. Three major themes were identified; Mental strategies, Understanding the complexity of Māori leadership, and Intergenerational development. This research affirms the importance of leadership for Māori communities, and in particular, the role of intergenerational knowledge-sharing as key for supporting rangatahi to be well.

year: 2024