Expression Of Human HMGB1 A Box In E.coli And Its Inhibitory Effects On Monocytes

Background and objectiveHigh mobility group box1 (HMGB1) protein which contains 215 amino acid sequence of highly conserved can widely expressed in mammalian cells. Because of its relative molecular weight is small(M<30000),it can rapidly migrated in the poly-acrylamide gel electrophoresis. HMGB1 consists three different functional areas, its N-terminal has two non-specific DNA binding domains:HMGB Abox and HMGB B box. Each of them is constituted by about 80 amino acid residues, which are rich in positively charged lysine. Its acidic C-terminal contains more than 30 consecutive aspartate residues and glutamate residues in order to complete the interaction with other protein and regulate the combinant between HMBG1 and DNA. HMGB1 protein distributed both inside and outside of the cell, it can play different biological effects according to its distribution. There are only two amino acid of HMGB 1 difference between human and mouse, the mouse of HMGB 1 deficient will die within a few hours after birth that can told us HMGB 1 protein plays an important role in the maintenance of the cell function. Intracellular HMGB 1 participate in the construction of nuclear body, the protein combined to DNA minor groove to change configuration of the DNA double helix, increase the interaction of affinity between transcription factors and DNA, while in DNA recombinant replication and repair the protein also play an important role. Extracellular HMGB1 possess cytokine properties, it is closely associated with cellular differentiation, migration, proliferation, apoptosis and inflammation reaction. Although it lacks signal peptide and can not through out of the endocytoplasmic reticulum and Golgi cellular organelle, HMGB1 can still be taken into the extracellular through two routes, the one route is HMGB1 release from necrotic but not apoptotic cell, the other route is inflammatory factor stimulate cell,these cells such as:macrophage, cell natural killer cell and so on. Extracellular HMGB 1 must be combined with the corresponding membrane receptor in order to play its biological effects. Receptor for advanced glycation end produces (RAGE) has high-affinity to HMGB1 and is the main receptor of HMGB1. After combining with RAGE, HMGB1 can initiate phosphorylation of MAPK, the intranuclear shift of nucleus transcription factor NF-κB and the activated protein-1 in order to start inflammartory and deteriorates inflammartory by making many other inflammatory factors like IL-1β, TNF-a, IL-8 be expressed and released highly. When the cell necrosis or damaged the HMGB1 of the nucleus can be released into the extracellular under the role of TNF, triggered monocyte-macrophage cells to secrete pro-inflammatory cytokines,while pro-inflammatory cytokines can also further promote the secretion of HMGB1,so a positive feedback loop was formed.In the latter part of inflammatory reaction,this positive feedback has an important role in the maintenance of inflammatory response such as SLE.HMGB1 and B box protein were added in cultured macrophages respectively, it was shown that after stimulated by both the levels of TNF-a generation positively increased, after the use of anti-B box protein antibody results showed that both TNF-a in generate levels were significantly lower, all was told us B box domain plays major role in inflammation-causing effects of HMGB1 and it alone can play. Similarly, in macrophages, after adding A box protein the production of TNF-a levels under HMGB1 or B box protein stimulated can significantly reduced. So we can say A box protein plays an important role in inhibiting inflammation reaction and has become an effective antagonist of HMGB1.To explore the therapeutic effect of HMGB1 A box in the SLE and other autoimmune diseases, we carried out the expression vector construction, recombinant protein purification and biological activity studies.Methods and results1. Cloning and analysis of human high mobility group box 1 A boxWe optimized the coding sequence of mature human HMGB1 according to the report of GenBank, adjust the codon adaptation index (CAI)makes the value of CAI is 0.95, increase the content of GC in coding sequences in order to extend the half-lives of mRNA, the secondary structure of mRNA also was removed, plasmid DNA pUC57-HMGB1 was synthetized. According to the optimized sequence of HMGB1 one pair of primers was designed. restriction sites of Ndel and Xhol were added to the upstream of forward and reverse primers respectively, amplified human HMGB1-A box fragment with Taq DNA polymerase. PCR product was separated by agarose gel electrophoresis, then gel extraction kit isolated purpose gene fragments. The purified PCR product was ligated with pMD19-T vector and then transformed to the competent of E.coilDH5α. The positive clones were primary screened with colony PCR and restriction map analysis. Using reverse and forward primers the positive clones were sequenced with ABI3100 DNA sequencer. The results showed that the cloned A box was similar to optimized sequence. Therefore, the recombinant HMGB 1 A box vector, pMD19-T/HMGB 1 A box, has been successful constructed.2. Expression of High mobility group boxl A box in E.coli and identification of recombinant proteinTo generate the recombinant prokaryotic expression vector, pMD19-T/HMGB 1 A box were digested with two restriction enzymes Nde1 and Xhol. The purpose fragment was separated with low-melting agrose gel electrophoresis and inserted into pQE-T7-2 that was digested with the same enzymes. The recombinant was transformed into E. coli DH5a and inoculated LB plate with 50ug/mL kanamycin, positive recombinants were selected with colony PCR. Positive recombinant colonies were selected and inoculated into LB liquid medium that contain 50ug/ml kanamycin,then we extracted the plasmid and transformed to the competent of E.coilBL21. selection of a single colony was inoculated into LB liquid medium that contain 50ug/ml kanamycin. The bacterium harboring pQE-HMGB 1 A box was grown to early log phase in LB medium containing kanamycin (50ug/mL) at 37℃, expression of the recombinant HMGB 1 A box was induced with IPTG at 37℃for 4 hours. The cells were collected by centrifugation and analysised by SDS-PAGE和Western blotting. The SDS-PAGE of the bacterium lysate shown that there was an additional band that seemed to be about 14 KDa identified with previously reported that recombinant HMGB 1 A box expressed in E.coli. Using Western blotting analysis, recombinant protein can responses with anti-HMGB 1 polyclonal antibody and anti His-tag polyclonal antibody shown that recombinant protein is the specific human HMGB1 A box protein.3. Human HMGB1 A box protein purification and biological activity assayCollecting biomass then lyased with supersonic wave in the ice bath, supernatant was collected. Precipitated which was washed by 0.5% triton X-100 shall be crude inclusion bodies. The products of supernatant after supersonic wave and inclusion body lysate were analysis by SDS-PAGE. The results showed that recombinant protein present in the supernatant, suggesting that HMGB1 A box with soluble forms expressed in the cytoplasm of strain. The purpose protein was purified by Ni2+-NTA chromatography then analysised by SDS-PAGE. It was showed that the purity of recombinant protein HMGB1 A box was over 90%.Immune complex (IC) was prepared with serum of patients with SLE and Cell necrosis fluid which was prepared according to literature in the ratio of 1:25. Experiment was done under two groups:U937cell+IC group, U937cell+IC+HMGB1 A box group, incubate at 37℃for 24h under the condition of 50ml/L CO2. Total RNA was isolated, the levels of IFN-γ,BAFF and TNF-a were detected by RT-PCR while GAPDH as an internal reference. Agarose gel electrophoresis of PCR products were scanned by gel image scanner and analyzed using the relative index which can reflect the variation of expression of BAFF, IFN-y and TNF-a. The results showed that HMGB1 A box protein which could effectively inhibit the secretion of mononuclear cells in BAFF, TNF-a and IFN-y have significant biological activity.Conclusions1) Using recombinant DNA technique, we have successfully cloned human mature HMGB1 A box gene, and its DNA sequence was the same with the optimized sequence.2) The recombinant prokaryotic expression vector pQE-HMGB1 Abox containing human HMGB1 A box has been constructed and can express in the stable manner.3) Some methods involved in the recombinant HMGB1 Abox expression and preparation and purification have investigated and optioned.4) The recombinant protein is the specific human HMGB1 A box protein which accounted for 40% of the total bacterial protein,5) The recombinant protein have significant biological activity.