Northern Kentucky University

Poster Title

Attempts at Constructing Novel Na, K ATPase/H, K ATPase Expression Vectors

Institution

Northern Kentucky University

Abstract

The Na, K ATPase and the H, K ATPase are two medically important ion transport membrane proteins. Their amino acid sequences are found to be very similar, but exact structures are not yet known. These two proteins are of interest to scientists because, although similar in sequence, they react to different classes of drugs and pump different ions. In order to determine something about the portion of the protein responsible for functional differences, three chimeras have been constructed containing a portion of each ion transporter. Attempts have been made to force expression of these chimeras in HeLa cells using of ouabain selection, but so far only one has worked. Another approach is being undertaken to allow expression of the other two chimeras in HeLa. Instead of using ouabain selection, as mentioned above, a second approach will be implemented using puromycin/neomycin resistance. Attempts are being made to construct plasmids containing both the chimera cDNA as well as a puro/neo resistance gene on a polycistronic DNA segment. In theory, both proteins will be expressed from the single mRNA via internal ribosomal entry sites. In order for this to be accomplished the chimeric cDNA must first be inserted into another vector, circumventing the need for a blunt end ligation, before it can be ligated into the expression vector. Once this construct is completed, it can be transfected into HeLa cells and ion transporter function can be studied.

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Attempts at Constructing Novel Na, K ATPase/H, K ATPase Expression Vectors

The Na, K ATPase and the H, K ATPase are two medically important ion transport membrane proteins. Their amino acid sequences are found to be very similar, but exact structures are not yet known. These two proteins are of interest to scientists because, although similar in sequence, they react to different classes of drugs and pump different ions. In order to determine something about the portion of the protein responsible for functional differences, three chimeras have been constructed containing a portion of each ion transporter. Attempts have been made to force expression of these chimeras in HeLa cells using of ouabain selection, but so far only one has worked. Another approach is being undertaken to allow expression of the other two chimeras in HeLa. Instead of using ouabain selection, as mentioned above, a second approach will be implemented using puromycin/neomycin resistance. Attempts are being made to construct plasmids containing both the chimera cDNA as well as a puro/neo resistance gene on a polycistronic DNA segment. In theory, both proteins will be expressed from the single mRNA via internal ribosomal entry sites. In order for this to be accomplished the chimeric cDNA must first be inserted into another vector, circumventing the need for a blunt end ligation, before it can be ligated into the expression vector. Once this construct is completed, it can be transfected into HeLa cells and ion transporter function can be studied.