Cloning, Expression And Functional Analysis Of Matrix Attachment Regions-Binding Protein Of Dunaliella Salina

Matrix attachment regions (MARs) or scaffold attachment regions (SARs) are DNA fragments that still bind to the nuclear matrices or scaffold after chromatin is digested with restriction endonucleases. Although there are no consensus sequences among different MARs, some sequence motifs, such as A-box and T-box, have been identified. It has been reported that MARs can make chromatin form loop-like structure and regulate gene expression. Several kinds of proteins specifically binding to MAR DNA sequences, which are called MAR-binding proteins (MARBPs), have been isolated and identified from various organisms, but not from Dnualiella salina.Dnualiella salina is a kind of unicellular green alga without cell wall, which is one of the most extremely halotolerant eukaryotes and can live in a wide range of NaCl concentration from 0.05 M to 5 M. As a bioreactor for producing pharmaceutical proteins, D. salina has its own advantage over other species. In this study, the MARBP cDNA was amplified from D. salina, then a recombinant prokaryotic expression vector containing the amplified cDNA was constructed, and transformed to E.coli BL21(DE3). After the recombinant protein was purified by Ni-NTA Spin Column, EMSA was performed to detect whether the recombinant protein would bind to MARs sequences in vitro. This study may lay a solid foundation for the study the mechanism of MAR regulation function.Part 1 Cloning and sequencing of D. salina MARBP1. Methods1.1 Extraction of total RNAs of D. salinaA total of RNAs was extracted using TRIzol from cells of D. salina.1.2 Cloning and sequencing of MARBP gene RT-PCR was performed to amplify the MARBP cDNA using one pair of primers designed according to the MARPB sequence of D. salina on GenBank. The resulting PCR products were cloned into T vector and then sequenced. Homology analysis was performed after sequencing.2. Results2.1 Extraction of a total of RNAs of D. salinaTotal RNAs extracted from D. salina was evaluated by 1 % agarose gel and OD₂₆₀/OD₂₈₀, which indicated that high quality RNA was isolated.2.2 Cloning and sequencing of MARBP geneThe obtained sequence was 1623 bp in length, which encoded 541 amino acids. Alignment analysis suggests that the isolated sequence shared high homology with the sequence on GenBank to which was submitted by us.Part 2 Construction and expression of prokaryotic expression vector1. Methods1.1 Construction of recombinant prokaryotic expression vectorAfter both the gene fragment and plasmid pET28a(+) were respectively digested with the same double restriction enzymes EcoR I and Nde I , the resulting fragment and linear plasmid were ligated by T₄ DNA ligase. The recombinant prokaryotic expression vector was identified by enzyme digestion and DNA sequencing.1.2 Expression of MARBP in E. coliThe expression vector pET-MBP was transformed to competent E. coli BL21, and one from the transformed colonies was cultured in LK medium. While the A₆₀₀ of culture reached 0.4 to 0.6 value, IPTG was add to the medium to induce the expression of the recombinant protein. Subsequently, the transformed E. coli cells were collected by centrifugation at 1000 rpm at 1, 2, 4, 6 and 8 h after induction. After 100μl 1×loading buffer was added to resuspend the cells collection, the resuspension was boiled at 100°C for 3 min. Electrophoresis was conducted on 10 % SDS-PAGE to detect the expression of the recombinant protein.1.3 Western blotting Total proteins of E. coli were run on 10 % SDS-PAGE and then transferred to NC membrane. The 6×his tag antibody was used to detect the expression of the recombinant protein.2. Results2.1 Construction of recombinant prokaryotic expression vectorThe results of restriction enzyme digestion and DNA sequencing revealed that the recombinant prokaryotic expression vector was successfully constructed.2.2 Expression and Western blotting analysis of MARBPThe result of SDS-PAGE showed that the recombinant MARBP was expressed at a high level after the induction of 0.02 mM IPTG at 37°C. A distinct band, approximate 75 KD, was expressed in the E. coli, which was estimated to be the recombinant MARBP. The result of Western blotting also showed that the recombinant protein was successfully expressed in E. coli cells.Part 3 Purification and functional analysis of expressed recombinant proteins1. Methods1.1 Purification of MARBPAfter the positive E coli cells collected by centrifugation were resuspended using binding buffer from the His bind purification kit, the resuspended cells were ruptured by high pressure under the condition of 1.5×10⁵ kPa for at least 1h. The following procedure for purification was performed according to the manufactory's instructions.1.2 Probe labeling at 3'endUse biotin 3' DNA labeling kit (PIERCE) to label the probes and to determine the efficiency of labeling.1.3 Electrophoretic mobility shift assays (EMSA)EMSA was used to determine the state of binding between DNA and protein. If DNA sequence can bind with proteins, the electrophoresis speed on SDS-PAGE gel will be changed and shift band appear. DNA sequences binding with and without protein can be distinguished using chemoluminescence.2 Results 2.1 purification of MARBPAfter the protein was purified by His bind purification kit (America), the recombinant protein accounted for more than 70% of the total proteins.2.2 Labeling of probes and EMSAAlthough the efficiency of labeling was not high, it was enough for the EMSA test. Result of EMSA was not as good as expected to be. The result of EMSA test reveled that the protein can't binding to the labeled probs.Part 4 Conclusions1. The cDNA sequence of D. salina is 1623 bp in length, encoding 541 amino acids; it shares 99% homology with the sequence on GenBank.2. The recombinant prokaryotic expression vector pET-MBP is successfully constructed. The recombinant MARBP protein, approximately 75KD, is high-efficiently expressed in E. coli, and the purified protein can not bind to the MARs as identified by EMSA.