Cloning And Prokaryotic Expression Of Zinc Finger Protein (ZFP580) Gene Isolated From Rat Cerebellum

Objective: To clone the full-length open reading frame cDNA sequence of Zinc Finger Protein (ZFP580, encoding 172 amino acids) gene from rat cerebellum. To clone and analyze the cDNA sequence of amino terminal encoding region (encoding 1-88 amino acids) and carboxyl terminal encoding region (encoding 89-172 amino acids) of ZFP580 gene. To construct prokaryotic fusion expression vectors and induces the expression of fusion protein GST-ZFP580.Methods: 1 Total RNA was extracted from rat cerebellum with Trizol solution.2 The specific primers were designed according to the cDNA encoding sequence of ZFP580 full-length open reading frame published by GenBank. The cDNA encoding sequences of ZFP580 full-length open reading frame, amino terminal encoding region and carboxyl terminal encoding region were obtained by RT-PCR. Sequence analysis of these fragments and its deduced amino acids were accomplished online at the National Center for Biotechnology Information servers and edited using the Primer5,Clustalx and DNAMAN programs.3 These cDNA fragments of ZFP580 gene were cloned into pMD18-T-Easy vector according to the instructions of the T-A clone kit and sequenced. Plasmids were extracted according to the instructions of plasmid extraction kit and digested respectively by restriction enzyme Xho I and Bgl II to select positive recombinants. The recombinant plasmids were named as pMD18-T-ZFP580, pMD18-T-C and pMD18-T-N respectively. The recombinant plasmids were sent to Takara Company to be sequenced by using the dideoxy chain-termination method.4 The pMD18-T-ZFP580 recombinant vector and pET-42a(+) fusion expression vector was digested by the Bgl II and Xho I respectively. The digested products were purified, recoverd and then linked by T4DNA ligase at 16°C for 1h. The recombinants were then transformed into E.coli DH5αcompetent cells. Several clones were randomly selected, then inoculated in 5mL LB liquid culture medium(containing Kan, 10μg/ml) respectively, and cultured 37°C at 220 rpm overnight. Plasmids were extracted according to the instructions of plasmid extraction kit and digested respectively by restriction enzyme Bgl II and Xho I to select positive recombinants. The cDNA encoding sequence of ZFP580 full-length open reading frame was sub-cloned into prokaryotic expressing plasmid pET-42a (+), generating pET-42a-ZFP580. Prokaryotic expression vector pET-42a-ZFP580 was sequenced by using the dideoxy chain-termination method and confirmed by reading both strands.5 Prokaryotic expression recombinant plasmids were transformed into E.coli BL21(DE3) to express the proteins. After the recombinant bacterium was induced with IPTG, the expressed recombinant protein was analyzed with SDS-PAGE.Results: 1 The total RNA product was examined by the electrophoresis of agarose gel containing formaldehyde. Results showed that there were two clear bands of 18S and 28S.The ratio A260/A280 and A260/A230 of RNA sample was all about 1.9, which demonstrated that RNA quantity and purity were well.It can be used in the following RT-PCR.2 The cDNA open reading frame sequence of ZFP580, amino terminal encoding region and carboxyl terminal encoding region were obtained by RT-PCR successfully. DNA sequencing results showed that the ZFP580 open reading frame sequence, the sequences of ZFP580 amino terminal encoding region and ZFP580 carboxyl terminal encoding region were exactly consistent with the sequence reported in GenBank.3 The results of double enzyme digestion with Bgl II and Xho I showed the recombinant plasmids construction of pMD18-T- ZFP580, pMD18-T-C and pMD18-T-N of ZFP580 gene is successful.4 The recombinant plasmids of pET-42a-ZFP580 and pET-42a- C were confirmed by restriction endonuclease digestion and DNA sequencing respectively. The result indicates that the recombinant plasmid of pET-42a-ZFP580 contains the correct open reading frame.5 The expression of the GST-ZFP580 fusion protein in E.coli BL21 (DE3) was analyzed by SDS-PAGE. No expected protein band was observed with induction of IPTG6 The sequence of the whole length cDNA of ZFP580 gene is 1100bp. It contains an open reading frame (519bp), encoding a protein consisted of 172 amino acids. The result of amino acids sequence analysis of zinc finger protein online demonstrated that the encoding protein of ZFP580 has two domains. The amino terminal region between amino acids 5 and 88 is also remarkable rich in Proline residues. The carboxyl terminal region between amino acids 94-172 includes three high conserved C2H2 Zinc finger motifs.Conclusion: 1 The cDNA open reading frame sequence of ZFP580, amino terminal encoding region and carboxyl terminal encoding region were successfully cloned. Those cDNA sequences are identical to the predicted ZFP580 gene of GenBank.2 The clone vectors of pMD18-T-N,pMD18-T-C,pMD18-T- ZFP580 and pET-42a-ZFP580 fusion expression vector were constructed successfully.3 No expected protein band was observed with induction of IPTG.4 The open reading frame of ZFP580 gene encodes a 172 amino acid protein containing two domains.