Choice of expression vector alters the localization of a human cellular protein.
The fusion of synthetic epitopes with proteins of interest is an important tool in the identification and characterization of recombinant proteins. Several mammalian expression vectors are commercially available containing unique identification tags or epitopes. These vectors offer a great advantage to researchers, as highly specific antibodies and purification resins against these specific epitopes are readily available. The tags facilitate immunologic assays and the purification of the recombinant proteins. The fusion of these epitopes with the recombinant proteins is not expected to alter the behavior of the protein of interest. In this report, we demonstrate that the mere expression of a cellular protein, hVIP/mov34, which we earlier identified as a cellular HIV-1 Vpr ligand, in two different vectors clearly altered its localization pattern in HeLa cells. Specifically, cloning of hVIP/mov34 in pcDNA3/HisA resulted in its nuclear localization, whereas the expression of this gene from a TOPO cloning expression vector, pcDNA3.1/V5/His, resulted in cytoplasmic expression. The native staining pattern of hVIP/mov34 using polyclonal antisera raised against hVIP/mov34 demonstrated cytoplasmic staining. During cloning, other leader sequences intended for targeting this protein into a cytoplasmic or a nuclear location were not fused to the actual ORF of this protein. Also, the amino acid sequence of the fusion region arising from cloning of hVIP/mov34 in both vectors does not match any reported NLS sequences. These results indicate that the choice of the expression vectors, as well as the position of synthetic epitopes, can significantly alter the behavior and the biology of recombinant proteins. This result suggests the need for a careful examination of these features when characterizing a newly identified protein.
Department of Pathology & Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
M P
MP
Ramanathan
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Ayyavoo
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DB
Weiner
D021581:Active Transport, Cell Nucleus / Q000235:genetics; D000595:Amino Acid Sequence; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D016336:Gene Products, vpr / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000096:biosynthesis / Q000138:chemical synthesis* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008969:Molecular Sequence Data; D011506:Proteins; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D016336:Gene Products, vpr; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D011506:Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C072621:Psmd7 protein, mouse