An open reading frame of the hyperthermophilic archaeon Aeropyrum pernix K1 APE2325, which composed of 474 bases, was cloned and expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL. The recombinant protein was puriWed by Ni-chelation aYnity chromatography. It showed a single band with a molecular mass of 18kDa in SDS–PAGE. The puriWed enzyme exhibited both phospholipase A2 and esterase activities with the optimal catalytic temperature at 90℃. The enzyme activity was Ca2+-independent. Kinetic analysis revealed its Km, kcat, and Vm for the p-nitrophenyl propionate substrate were 103μM, 39s-1, and 249μmol/min/mg, respectively. The recombinant protein was thermostable and its half-life at 100℃ was about 1h.

应用悬滴汽相扩散法，用PEG4000作沉淀剂，获得了嗜热古菌Aeropyrum pernix 酰基氨基酸释放酶APE1547的一种新晶型，这种新晶型属于正交晶系，晶胞参数α=6.399nm, b=10.439nm. C=16.953nm、晶体空间群属于P212121。单位晶胞中每个晶体不对称单位含2个分子，Matthews常数为2.2×10-3nm3·-1，溶剂含量为43%，晶体衍射的最高分辨率达0.27nm，与以前获得的三斜晶系的晶体相比，这咱新晶型更适合于结构分析，可以加快结构解析的过程。

极端酶具有超常的生物学稳定性，能够在极端温度、pH、压力和离子强度下表现出生物学活性，因此极端酶为生物催化和生物转化提供了良机。新的极端物种的发现、基因组序列的确定及基因工程技术的应用，加快了发现和制备新酶的进程。蛋白质工程和定向进化技术进一步改善酶的活性和特异性，促进了极端酶的工业应用。对极端酶的研究加深了人们对酶稳定性机制的理解，丰富了分子进化理论,

A histone-like gene, PHS051 from hyperthermophilic archaeon Pyrococcus horikoshii OT3 strain, was cloned, sequenced, and expressed in Escherichia coli. The recombinant histone, HPhA, encodes a protein of 70 amino acids with a molecular weight of 7868 Da. Amino acid sequence analysis of HPhA showed high homology with other archaeal histones and eukaryal core histones. The HPhA was purified to homogeneity by heat precipitation and affinity chromatography. Gel electrophoresis mobility shift assays demonstrate that the purified HPhA has high affinity to DNA. The complex of the HPhA and DNA allows DNA to be protected from cleavage by the restriction enzyme TaqI at 65℃. Circular dichroism spectra reveal that the conformation of the recombinant histone HPhA becomes looser when temperatures increase from 25 to 90℃. The HPhA has inherited a remarkable thermostability especially in the presence of 1M KCl and retained DNA binding activity at extreme temperature, which is consistent with our previous report about its structure stability analyzed by X-ray crystallography.

HPhA, a recombinant histone-like protein from Pyrococcus horikoshii OT3 strain, has compacting activity with DNA as previously reported. The extreme stability and DNA packaging activity of the HPhA make it a candidate as a DNA carrier. Here, the plasmid DNA-HPhA complexes were fully characterized by gel retardation assay and DNase resistance assay. It was further proved that HPhA has in vitro DNA transfection activity. HPhA-mediated transfection efficiency was dependent on the mass ratio of HPhA to DNA, the incubation time and the presence of calcium. A protocol for HPhA-mediated transfection in vitro was established to improve transfection efficiency. The optimal mass ratio of HPhA to DNAwas 6:1, and the incubation time required for the DNA-HPhA complex to be in contact with the cell was 4 h. In addition, the presence of 2 mM CaCl2 in the cell culture medium was required for efficient transfection. Serum did not show inhibition of HPhA-mediated transfection. Most importantly, the cytotoxicity of HPhA is lower than that of commonly used cationic liposome-based gene delivery systems, and HPhA-mediated transfection in NIH 3T3, HEK 293, HL-7702, HepG2 and Cos 7 cell lines in vitro has a higher efficiency and reproducibility. These results demonstrate that the HPhA is a new, potentially widely applicable and highly efficient gene carrier.