Characterization of the LEB-3 gene expressed during erythroid terminal differentiation
Moskalik, Karen L
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The objectives of this research were to identify candidate genes for a previously isolated partial cDNA that is differentially expressed during erythroid terminal differentiation (LEB-3), and to create LEB-3 recombinant peptides for use in generating polyclonal antibodies in rabbits. The LEB-3 clone was subjected to BLAST analysis against the mouse genome. A candidate gene sequence was identified and used to design LEB-3 primers that were used in RT-PCR to amplify candidate gene cDNAs from erythroblasts. The cDNAs (LEB3-1 and 3-2) were cloned, sequenced and compared to the candidate gene sequence in Genbank and showed 99% homology. LEB-3-1 mRNA increased nearly 11 fold during erythroid terminal differentiation. LEB-3-1 mRNA was also expressed in cultured murine erythroleukemia (MEL) cells and in the testes of mice. Production of LEB-3-1 recombinant protein was achieved with the pBAD-His expression vector system and it was purified with a nickel affinity column under denaturing conditions.