| All organisms can be divided into one of the three major domains:bacteria, archaea and eukarya on the basis of small subunit ribosomal RNAsequences, with the deepest phylogenetic branch separating the bacteriallineage from archaeal/eukaryal lineage. Members of the archaea display whatare generally considered to be both bacterial and eukaryal features, whichindicates a possible evolutionary relationship between eukarya and archaea.An impressive example of eukaryal-associated feature is the archaeal histones.Histones, which are highly basic DNA binding proteins, are abundant andconserved in eukaryotic cells. Histone-like proteins are also conserved inarchaea. Studies show that the primary sequences, tertiary structures, anddimer formation of archaeal histones are conserved in the eukaryalnucleosome core histones H2A, H2B, H3, and H4. Many hyperthermophilic archaea have been isolated from deep-seahyperthermal environments and they can grow at temperatures near 100 .Pyrococcus horikoshii OT3 strain is a hyperthermophilic archaeon with theoptimum growth temperature at 98 . The genome sequence of this organismhas been completed at the National Institute of Technology and Evaluation(Tokyo, Japan). Two open reading frames PHS051 (HPhA) and PHS046(HPhB) were putative archaeal histones. We studied PHS051 (GenBankAccession No. AP000007). According to gene sequence and the analysis ofbioinformation we presume that PHS051 gene can encode a kind ofhistone-like protein. At first Pyrococcus horikoshii OT3 strain was cultured,and the genome DNA was obtained by molecular biology methods. ThePHS051 gene was fished from the genome DNA by PCR, and successfullycloned to plasmid pET11a. The recombinant plasmid pET11a-His wastransformed to E. coli BL21-Codon Plus. However, the expressive quantity ofHPhA is low. The reason is because the usage of the arginine codons AGA andAGG are much higher in Pyrococcus furiosus than that in E. coli, and theHPhA contains large amounts of arginine (7%). Using the molecular biologicaltechnique, modifying the traditional technique of PCR fixed point mutationand according to the sequence for the PHS051 gene sequence and the table forpartial codons of E. coli, this study has desighed a pair of simple chains whichcontain a mutated point. The entire length is 115nts. At the end of 3', 20nts arecomplementary. We denaturalize the simple chains of DNA at 94 ,renaturalize them at 45 to unite them and then extend them at 72 . Thus, weproduce histone gene with the mutated point. With this as the template, wedesign the corresponding primers and end up with the massive histone mutatedgene. Subsequently, the mutated gene was cloned to plasmid pET11a andexpressed in E. coli BL21-Codon Plus. The expression quantity ofrecombinant HPhA was obviously increased. The recombinant E. coli was cultured ~0.6 (OD600) in 2YT medium withampicillin. After IPTG induction the E. coli was continued to culture at 25for 12 h. Cells were collected by centrifugation. The obtained cells were lysedby sonication. After heat treatment, DNase treatment and centrifugation, theobtained supernatant was used for further protein purification by PD-10 andheparin affinity chromatography. The purity of the obtained HPhA is beyond90% by the analysis of Tricine-SDS-PAGE and HPLC. The MALDI-TOFmass spectrum showed that the molecular weight of HPhA is 7860.9Da. Theisoelectric point of HPhA is pH 9.05 by the isoelectrofocusing experiment.The result of analysis of amino acid composition of the recombinant HPhA iscoincident with theoretical value. Gel electrophoresis mobility shift assays demonstrate that thepurified HPhA has high affinity to DNA. The HPhA can increase linearplasmid electrophoresis mobilities with proper HPhA/DNA mass ratios.The complex of the HPhA and DNA allows DNA to be protected fromcleavage by the restriction enzyme Taq at 65 . Circular dichroismspectra reveal that the conformation of the recombinant... |
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