Erythroid Transcription Factor Fklf Expression, Purification And Preliminary Study

The programmed expression of globin genes is tissue and developmental stage specific. In humans, five -like globin gemes (, A, G, , and ) from a cluster on the short arm of chromosome 11, and their expression is characterized by tow major switches initially from embryonic (s) to fetal (A and G) and subsequently to adult (, and ) globin gene expression. Although a number of cis-acting elements of globin genes and corresponding trans-acting factors have been identified, the precise molecular mechanisms of globin gene regulation are still unvlear. Especially limited is the information on the trans-acting factors involved in the developmental control of fetal and embryonic globin genes.Among trans-acting factors involved in regulation of p-like globin genes, those Kr ppel like factors with Cys2-His2 zinc finger are intensely investigated. Sp1, a ubiquitous Kriippel like zinc finger protein, and EKLF, an erythroid tissue-specific Kriippel like zinc finger protein, are well characterized. Spl is known to interact with the , and -globin gene CACCC boxes; and EKLF binds to the p-globin gene CACCC box, and plays critical role in the expression of P-globin gene.In recent years, two novel Kriippel factors are identified subsequently. According to current studies, we know FKLF is consisted of 512 amino acid residues, and has three contiguous zinc fingers near carboxyl end, which are identity to the zinc finger structure of Spl, EKLF and other KLF family's members. According to the amino acid homologue of zinc finger, FKLF belongs to the third sub-family of Kriippel like zinc finger factors. FKLF is characterized by the presence of two acidic and two proline-rich regions in the long amino terninal domain. The two acidic regions constitute the key domain of the frww-activation function. FKLF mainly expresses in the erythoid cells, and may be a activator of embryonic and fetal globin gene expression in vivo. Until now, no studies on its prokaryotic expression and protein purification have been found.In the present paper, the FKLF encoding sequence was amplified frompBS/FKLF by PCR, and was inserted into the prokaryoic expression plasmid pET32a(+). Then, the recombinant plasmid was transformed into E. coli BL21(DE3). The protein FKLF was expressed as inclusion bodies in E. coli after 3 hours'IPTG induction, and was seperated from total bacterial protein on 8% polyacrylamide gel. After purification and concentration, the protein FKLF reached to 3.5 mg per liter culture. Using the purified FKLF as antigen, the house rabbit and Balb/C mouse were immunized to get anti FKLF serum. The efficiency of antibody was 1:800.Moreover, we constructed three eukaryotic expression plasmids pIRES-EGFP/FKLF, pcDNA3/FKLF and pIRES-pac/FKLF. Our study demonstrated that the expression of FKLF was obviously higher in K562 cells containing the three plasmids than that in the control cells. The result made foundation on further investigation of the role of FKLF in embryonic and fetal globin gene expression, and provided possibility to obtain stable expresssion cell lines.Furthermore, we studied whether hydroxyurea, a kind of inducer of fetal hemoglobin, had effect on endogenous FKLF expression in K562 and HEL cells. As a result, it showed that hydroxyurea didn't up-regulate the expression of endogenous FKLF in the transcriptional level. And also no positive bands were detected in the translation level by western blotting. We proposed that there would have other transcriptional factors affected the expression of FKLF.