Addition of a Histidine Tag onto Nucleobindin and Protein Expression in SF21 Insect Cells
Nucleobindin (Nuc) is a transcription factor-like protein that causes thymic apoptosis when injected into mice and is associated with the pathogenesis of systemic Lupus erythematosis. Nuc interacts with Prostaglandin Endoperoxidase H Synthase (PGHS), which catalyzes the transformation of arachidonic acid into the prostaglandins. Although this interaction is likely to be physiologically important little is known about the effect of Nuc binding on PGHS structure and function. The general goal of this project was to obtain the crystal structure of Nucleobindin alone and in complex with PGHS. In order to do this it is important to have an efficient way of purifying this protein. Thus, we set out to engineer a Histidine addition (His-tag) onto Nuc, which would allow for a more facile purification of Nuc through the Ni/NTA column. We designed a primer with nucleotides coding for the 6x His-tag and used Gene Editor kit (Promega) to incorporate it into the Nuc gene. We confirmed that the His-tag was present on the Nuc gene by sequencing it through the 3' end. Also, we have made a 6x His:Nuc baculovirus construct for expression in SF21 insect cells, but have not yet expressed this protein in these cells. After His-tagged Nuc is expressed it can be more easily purified through the Ni/NTA column. With gram quantities of pure protein readily available we will be able to begin crystallization trials to determine the tertiary and quartinary structure of Nuc and Nuc in conjunction with PGHS.
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