RECOMBINANT EXPRESSION OF TYPE IV P-TYPE ATPASES AND CONSTRUCTION OF GENE TARGETING VECTORS
Open Access
- Author:
- Deering , Rebecca Sue
- Graduate Program:
- Genetics
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- None
- Committee Members:
- Robert Allen Schlegel, Thesis Advisor/Co-Advisor
Robert Allen Schlegel, Thesis Advisor/Co-Advisor - Keywords:
- phosphatidylserine
ATP8A1
gene targeting vectors
baculovirus protein expression
ATP8A2
type IV P-type ATPases
ATP9A
ATP9B - Abstract:
- The distribution of phospholipids in the plasma membrane and the membranes of intracellular organelles in eukaryotic cells is asymmetric. Sphingomyelin and phosphatidylcholine are present in the outer layer of the plasma membrane or the lumenal side of intracellular membranes, and phosphatidylserine and phosphatidylethanolamine are restricted to the cytosolic side of membranes. The maintenance of phospholipid asymmetry is an energy-dependent process. The type IV Ptype ATPases are classified as putative amphipath transporters. Indirect evidence suggests that the mammalian class 1 transporters 1a (ATP8A2), 1c (ATP8B1), and 1e (ATP8B3) transport, and thereby restrict phosphatidylserine to the cytosolic side of membranes. To investigate if all class 1 type IV P-type ATPases transport phosphatidylserine and whether enzymes of other classes of type IV P-type ATPases also transport phosphatidylserine, selected mammalian genes were expressed in Spodoptera frugiperda insect cells using a baculovirus expression system, which produces large enough quantities of recombinant proteins for biochemical analyses of substrate specificity. Based on similarity to the 1a gene, the protein encoded by the 1b gene (67% identical; 82% similar) is the most likely candidate to transport the same amphipathic molecule, phosphatidylserine, and the protein encoded by the 2b gene (29% identical; 48% similar) is the least likely. If the 2b protein also transports phosphatidylserine this will suggest that all type IV P-type ATPases transport phosphatidylserine. Accordingly, using the pFastBac HTb vector, recombinant 2b protein was expressed with an N-terminal 6x histidine tag to allow detection of the protein using a probe specific to consecutive histidines. Both the histidine probe and a monoclonal antibody directed against 2b detected a protein of the same molecular weight as recombinant 2b, but in both baculovirus-infected and uninfected cells, precluding specific detection of recombinant 2b protein. 1b was initially expressed with a 6x N-terminal histidine tag using the start codon within the pFastBac HTb vector. Detection of recombinant 1b protein expressed from this vector was not consistent due to low expression of the recombinant protein or due to improper folding of the protein resulting in inaccessibility of the histidine tag. To increase expression of the 1b protein the 1b expression vector was reconstructed using the pFastBac 1 vector. An engineered polyribosomal binding site was placed upstream of the 1b initiation codon to increase protein expression and to allow the use of the 1b initiation codon, reducing the addition of non-functional amino acids. To decrease the chances of the recombinant protein being incorrectly folded, the 6x histidine tag was moved to the 3' end of the 1b ORF and engineered to allow protein detection and purification of the recombinant protein using nickel-nitrilotriacetic acid agarose. Higher levels of recombinant 1b protein expression were achieved using this vector. However, attempts to purify the recombinant 1b protein were unsuccessful, perhaps due to insufficient solubility of the protein. After sufficient quantities of soluble recombinant 1b and 2b proteins are purified, biochemical assays will be performed to determine if phosphatidylserine stimulates 1b and/or 2b ATPase activity. Briefly, in the presence of Mg2+, ATP will phosphorylate the iv purified enzyme at a conserved aspartate residue in the catalytic site forming a phosphoenzyme intermediate. Upon addition of the correct substrate (e.g., phosphatidylserine), the enzyme will dephosphorylate releasing inorganic phosphate. The amount of phosphate released is indicative of enzyme activity. If stimulation is specific, the phosphoenzyme will only dephosphorylate in the presence of the sn-1,2 stereoisomer. To study the physiological functions of type IV P-type ATPases, gene targeting vectors were constructed in order to knock out the 1a and 2b genes. Similar to the protein expression studies, these two genes were chosen because they are least similar to each other. Because a gene targeting vector should replace coding sequence within the 5' end of a gene to ensure a non-functional protein is produced after homologous recombination in embryonic stem cells. Southern analysis probes IIb and 2bgenom were designed and used to identify restriction fragments from BAC 23170 containing exons 1, 2, 3, or 4 of the 2b gene, and the identified fragments inserted into pBluescript vectors. Three plasmids were obtained. Two plasmids, POO and PW, contain sequence that encodes exon 4 and a portion of transmembrane domain 2. PH#6 contains sequence encoding the translational start codon, transmembrane domain 1, exon 1, exon 2, and exon 3 and will be used to generate the targeting vector. In constructing a gene targeting vector, a positive selection cassette, such as the neomycin resistance cassette, is used to replace a coding portion of the gene of interest and allow identification of homologously recombinant embryonic stem cells. A previously constructed 1a gene targeting vector contained a neomycin positive selection cassette that replaced 2,473 bp of the 1a ORF, including the coding sequence for transmembrane domain 1, 30 bp of exon 2, exon 3, and 22 bp of exon 4; a 3-frame stop adjacent to the 5' end of the neomycin resistance cassette; and 5' and 3' homology regions. The 3-frame stop ensures that functional protein will not be produced. The diptheria toxin A negative selection cassette was placed at the end of the 3' homology region of this vector to allow identification of only homologously recombinant embryonic stem cells. The revised construct was linearized and given to the PSU Transgenic Facility to obtain recombinant embryonic stem cell clones that could be used to generate a 1a knockout mouse. A 5' hybridization probe containing exon 5 sequence and a 3' hybridization probe containing intron 3 sequence were generated to enable verification of homologously recombinant embryonic stem cells via Southern analysis of mouse tail DNA. Mutations within the class 1 gene ATP8B1, which is 47% identical and 65% similar to the 1a gene, cause liver disease in humans, indicating that other type IV P-type ATPases are not able to compensate for its loss of function. Therefore, the 1a and 2b knockout mice will be examined physically and histologically to determine if the loss of function of either gene has an effect on the health of the mice. Since 1a is most highly expressed in the brain and lungs, and 2b in the testes, we may expect the 1a knockout mouse to have a central nervous system defect and the 2b knockout mouse to have fertility problems.