中国林蛙核糖核酸酶Rdrlec新基因的原核可溶表达

来源于蛙属的核糖核酸酶由于具有显著的抗肿瘤活性而备受关注,Rdrlec是从中国林蛙基因组中克隆得到的核糖核酸酶新基因。获得大量高纯度野生型重组蛋白是研究其功能的基础。按照大肠杆菌偏好的密码子人工合成Rdrlec基因,通过EcoR I和Hind III位点插入到表达载体pET-32a(+)中构建pET32-Rdrlec重组表达质粒,转化到Escherichia coli BL21(DE3)中,0.4 mmol/L IPTG 30℃诱导6 h后,融合蛋白主要以可溶形式表达,经过Ni-NTA亲和纯化和Sephadex G75层析纯化,得到电泳纯融合蛋白。肠激酶切割后得到Rdrlec野生型重组蛋白,具有降解RNA的酶活性,证明分子的空间结构已经正确形成。Rdrlec野生型重组蛋白表达成功,为后续蛋白结构与功能的研究以及进一步的开发应用提供了原料。     

福氏志贺菌硫氧还过氧化物酶的表达、纯化、活性检测及晶体生长的初步研究

Sf2523蛋白属于硫氧还蛋白过氧化物酶(Prx)家族,在有氧代谢过程中消除活性氧,起到保护生命大分子的重要作用.通过构建原核表达体系,可溶性表达并纯化了Sf2523酶蛋白,并对蛋白进行了过氧化物酶活性检测,证明其依然具有天然活性,酶的核心结构并未发生变化.由分子排阻色谱结果发现,酶蛋白体内和体外聚集状态不同,离体蛋白聚集状态不稳定,趋于二体化.将纯化的单体蛋白即时进行了结晶实验,初筛长出了针状晶体.通过进一步切除标签蛋白进行优化处理,最终得到了均匀的三维单晶.     

鸡氨肽酶N的高效可溶性表达及生物学功能分析

本研究旨为克隆鸡氨肽酶N(chAPN)基因,高效表达可溶性目的蛋白,并测定其生物学功能。应用RT-PCR方法从鸡胚肾细胞中克隆chAPN的基因片段,经测序鉴定后再克隆至原核表达载体pCOLD-TF,构建重组原核表达质粒pCOLD-TF-chAPN,在大肠杆菌BL21(DE3)中经不同条件诱导表达目的蛋白;利用镍柱亲和层析法纯化可溶性蛋白,并进行SDS-PAGE、Western blotting鉴定;Leu-PNA酶促反应和ELISA等方法检测目的蛋白生物学功能。结果显示,重组质粒pCOLD-TF-chAPN在大肠杆菌中以可溶形式高效表达;酶促反应及ELISA结果显示该蛋白具有酶活性,可结合传染性支气管炎病毒(IBV),并表现为剂量依赖性。这为今后研究chAPN的酶活性、作为IBV受体及抗病毒功能奠定了实验基础。     

棉铃虫单核衣壳核多角体病毒解螺旋酶基因的序列分析

对棉铃虫单核衣壳核多角体病毒（Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus,HaSNPV）基因组中EcoR I-N片段进行序列分析,获得了完整的解螺旋酶基因（hel）,其开放阅读框大小为3762bp,编码一个分子量为146kD的蛋白质。在hel起始密码子ATG上游50位有强晚期启动子转录起始信号ATAAG,在－112位和－189位存在两个TATA box,但未发现早期转录信号CAGT。其在终止密码子下游第12位有一PolyA终止信号AATAAA。在其它真核或原解螺旋酶中存在的7个保守基元（Ⅰ、Ⅰa、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ）,只有5个（Ⅰ、Ⅰa、Ⅱ、Ⅲ、Ⅳ）在杆状病毒中保守。同源性比较发现,HaSNPV解螺旋酶的氨基酸序列与甜菜夜蛾核多角体病毒（Spodoptera exigus MNPV,SeMNPV）的解螺旋酶具有最高的同源性（66％）,与Xestia c-nigrum颗粒体病毒（XcGV）解螺旋酶的同源性最低（43％）。HaSNPV解螺旋酶基因是第一个报道的单粒包埋核多角体病毒的解螺旋酶基因。     

解螺旋酶RECQL5的功能及其在肿瘤中作用的研究进展

目前,研究已在小鼠和人类中发现5个Rec Q基因家族成员,分别是Recql1、BLM、WRN、Recql4和RECQL5。其中,RECQL5具有抑制姐妹染色体交换、抑制同源重组修复、阻止双链DNA断裂以及与TOPⅡα的协同作用等功能。最近研究在缺乏RECQL5的Apcmin小鼠小肠和结肠中均发现有肿瘤生长,提示RECQL5蛋白与肿瘤的发生密切相关。此外,RECQL5可能是影响喜树碱(CPT)治疗结直肠癌效果的主要因素,并可能是以伊立替康为基础药物治疗结肠癌效果评估的潜在生物标记物。本文就解螺旋酶RECQL5的功能及其在肿瘤中作用的研究进展进行综述。     

糖化酶酶解米渣纯化米蛋白的工艺

研究了糖化酶酶解米渣纯化米蛋白的实验条件:液固比、酶解时间、pH、温度和酶量。通过正交实验优化了酶解主要条件,得到糖化酶水解米渣最佳条件:液固比5:1,时间3 h,pH 4.0,温度65℃和酶量30 U.g-1。在最佳条件下实验,米蛋白的提取率为81.3%,纯度为76.8%。     

圆红冬孢酵母新颖脂肪酸合酶的重组表达、纯化与活性检测

脂肪酸合酶(Fatty acid synthase,FAS)催化乙酰辅酶A和丙二酸单酰辅酶A反应生成脂肪酸,是油脂合成代谢途径中最重要的酶之一。在高产油脂的圆红冬孢酵母Rhodosporidium toruloides中发现了一种新颖的FAS,它含两个亚基,与其他物种的FAS相比,具有独特的结构域组成,尤其是含两个酰基载体蛋白(ACP)结构域。由于ACP在脂肪酸合成反应中起辅因子作用,推测多个ACP有利于提高FAS的催化活性,为研究该FAS的生物化学和结构特征,构建了表达FAS两个亚基的载体,并转化大肠杆菌Escherichia coli BL21(DE3),含pET22b-FAS1和pET24-FAS2质粒的重组菌株ZWE06可同时高表达两个亚基,经硫酸铵沉淀、蔗糖密度梯度离心和阴离子交换层析纯化,得到的重组FAS比活力达到548 mU/mg。纯化的FAS复合物可用于后续酶动力学和蛋白结构研究,且表达与纯化方法的建立对研究其他ACP的功能具有参考价值。     

人转录因子hASH4的表达纯化及其DNA结合活性

hASH4蛋白所属的HLH转录因子家族在调节基因表达,调控细胞周期,决定细胞分化中起了重要作用。有研究表明hASH4蛋白可能与皮肤的分化发育有着密切的关系,但具体机制不明。成功构建了pET28b-hASH4表达质粒,并在大肠杆菌BL21(DE3)中诱导表达。经过对温度、时间、IPTG浓度等表达条件的优化,确定在37℃下1 mmol/L IPTG诱导表达4 h可达到最佳表达效果,并通过亲和层析和弱阳离子交换层析纯化蛋白,得到了电泳纯的目的蛋白。通过非放射性凝胶滞留试验发现hASH4蛋白单体只具有非特异性的DNA结合活性,而不具有特异性的DNA结合活性,推进其在体内的转录因子功能可能需先形成异源二聚体或多聚体才能进一步特异性结合DNA进而作用于下游基因。研究结果为hASH4蛋白的功能和作用方式提供了线索,并为其进一步的结晶条件筛选、晶体结构解析和功能研究奠定基础。     

具有蛋白酶及解旋酶活性的HCV-NS3重组蛋白的纯化及活性分析

为深入探讨HCV-NS3蛋白的酶动力学性质,制备了具有蛋白酶及解旋酶活性的HCVNS3重组蛋白。利用PCR扩增HCV非结构基因NS3,插入pPIC9,测序分析。携带NS3基因的重组质粒(pPIC9-NS3)转化毕氏酵母菌菌株GS115,甲醇诱导表达NS3蛋白。重组蛋白首先采用Hitrap chelating柱进行亲和分离,之后使用Mono S HR柱进一步纯化。对纯化后的NS3重组蛋白的酶活性进行分析,结果表明,获得的重组蛋白分别具有蛋白酶及解旋酶活性。本研究为深入探讨NS3编码酶的功能和开发抗病毒药物创造条件。     

棉铃虫单核衣壳核多角体病毒解螺旋酶基因的序列分析

对棉铃虫单核衣壳核多角体病毒(Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus,HaSNPV)基因组中EcoRI-N片段进行序列分析,获得了完整的解螺旋酶基因(hel),其开放阅读框大小为3 762bp,编码一个分子量为146kD的蛋白质.在hel起始密码子ATG上游50位有强晚期启动子转录起始信号ATAAG,在-112位和-189位存在两个TATA box,但未发现早期转录信号CAGT.其在终止密码子下游第12位有一PolyA终止信号AATAAA.在其它真核或原核解螺旋酶中存在的7个保守基元(I、Ia、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ),只有5个(I、Ia、Ⅱ、Ⅲ、Ⅳ)在杆状病毒中保守.同源性比较发现,HaSNPV解螺旋酶的氨基酸序列与甜菜夜蛾核多角体病毒(Spodoptera exigue MNPV,SeMNPV)的解螺旋酶具有最高的同源性(66%),与Xestia c-nigrum颗粒体病毒(XcGV)解螺旋酶的同源性最低(43%).HaSNPV解螺旋酶基因是第一个报道的单粒包埋核多角体病毒的解螺旋酶基因.     

Determination of the biochemical properties of full-length human PIF1 ATPase

The PIF1 helicase family performs many cellular functions. To better understand the functions of the human PIF1 helicase, we characterized the biochemical properties of its ATPase. PIF1 is very sensitive to temperature, whereas it is not affected by pH, and the ATPase activity of human PIF1 is dependent on the divalent cations Mg²⁺ and Mn²⁺ but not Ca²⁺ and Zn²⁺. Inhibition was observed when single-stranded DNA was coated with RPA or SSB. Moreover, the ATPase activity of PIF1 proportionally decreased with decreasing oligonucleotide length due to a decreased binding ability. A minimum of 10 oligonucleotide bases are required for PIF1 binding and the hydrolysis of ATP. The analysis of the biochemical properties of PIF1 together with numerous genetic observations should aid in the understanding of its cellular functions.     

Characterization of ATPase activity of recombinant human Pif1

Saccharomyces cerevisiae Piflp helicase is the founding member of the Pifl subfamily that isconserved from yeast to human.The potential human homolog of the yeast PIFI gene has been cloned fromthe cDNA library of the Hek293 cell line.Here,we described a purification procedure of glutathione S-transferase(GST)-fused N terminal truncated human Pifl protein(hPif1ΔN)from yeast and characterizedthe enzymatic kinetics of its ATP hydrolysis activity.The ATPase activity of human Pif1 is dependent ondivalent cation,such as Mg~(2 ),Ca~(2 )and single-stranded DNA.Km for ATP for the ATPase activity isapproximately 200 μM.As the ATPase activity is essential for hPifl's helicase activity,these results willfacilitate the further investigation on hPif1.     

Human PIF Helicase is Cell Cycle Regulated and Associates with Telomerase

The evolutionarily conserved PIF1 DNA helicase family is important for the maintenance of genome stability in the yeast, Saccharomyces cerevisiae. There are two PIF1 family helicases in S. cerevisiae, Pif1p and Rrm3p that both possess 5’→3’ DNA helicase activity but maintain unique functions in telomerase regulation and semi-conservative DNA replication. Database analysis shows that the PIF1 helicase family is represented by a single homologue in higher eukaryotes. To analyze the function of PIF1 homologues in mammals, we cloned the full length human PIF (hPIF) cDNA. Comparison of hPIF with its S. cerevisiae homologues showed that human PIF is equally similar to Pif1p and Rrm3p. Human PIF1 was expressed at low levels in a variety of tissues and immunofluorescence analysis showed that ectopic hPIF1 was localized to nuclear foci. hPIF was expressed in late S/G2 phase of the cell cycle and this cell cycle regulated abundance was conferred by both cell cycle regulated mRNA accumulation and ubiquitin-mediated degradation. Furthermore, hPIF is likely a target of the anaphase promoting complex/cyclosome as its abundance was decreased when an activator of the APC/C was over-expressed. Finally, antibodies against hPIF immunoprecipitated telomerase activity from human cell lines, and we have observed a physical interaction between hPIF and the catalytic subunit of telomerase, hTERT. Our data suggest that human PIF, like S. cerevisiae Pif1p, plays a role in telomerase regulation.     

Biochemical analysis of human PIF1 helicase and functions of its N-terminal domain

The evolutionary conserved PIF1 DNA helicase family appears to have largely nonoverlapping cellular functions. To better understand the functions of human PIF1, we investigated biochemical properties of this protein. Analysis of single-stranded (ss) DNA-dependent ATPase activity revealed nonstructural ssDNA to greatly stimulate ATPase activity due to a high affinity for PIF1, even though PIF1 preferentially unwinds forked substrates. This suggests that PIF1 needs a ssDNA region for loading and a forked structure for translocation entrance into a double strand region. Deletion analysis demonstrated novel functions of a unique N-terminal portion, named the PIF1 N-terminal (PINT) domain. When the PINT domain was truncated, apparent affinity for ssDNA and unwinding activity were much reduced, even though the maximum velocity of ATPase activity and K_m value for ATP were not affected. We suggest that the PINT domain contributes to enhancing the interaction with ssDNA through intrinsic binding activity. In addition, we found DNA strand-annealing activity, also residing in the PINT domain. Notably, the unwinding and annealing activities were inhibited by replication protein A. These results suggest that the functions of PIF1 might be restricted with particular situations and DNA structures.     

A single-stranded DNA binding protein required for mitochondrial DNA replication in S. cerevisiae is homologous to E. coli SSB.

It has previously been shown that the mitochondrial DNA (mtDNA) of Saccharomyces cerevisiae becomes thermosensitive due to the inactivation of the mitochondrial DNA helicase gene, PIF1. A suppressor of this thermosensitive phenotype was isolated from a wild-type plasmid library by transforming a pif1 null strain to growth on glycerol at the non-permissive temperature. This suppressor is a nuclear gene encoding a 135 amino acid protein that is itself essential for mtDNA replication; cells lacking this gene are totally devoid of mtDNA. We therefore named this gene RIM1 for replication in mitochondria. The primary structure of the RIM1 protein is homologous to the single-stranded DNA binding protein (SSB) from Escherichia coli and to the mitochondrial SSB from Xenopus laevis. The mature RIM1 gene product has been purified from yeast extracts using a DNA unwinding assay dependent upon the DNA helicase activity of SV40 T-antigen. Direct amino acid sequencing of the protein reveals that RIM1 is a previously uncharacterized SSB. Antibodies against this purified protein localize RIM1 to mitochondria. The SSB encoded by RIM1 is therefore an essential component of the yeast mtDNA replication apparatus.     

大肠杆菌RecQ解旋酶的表达纯化和生物学活性研究

RecQ家族解旋酶是DNA解旋酶中高度保守的一个重要家族,在维持染色体的稳定性中起着重要的作用.人类RecQ家族解旋酶突变会导致几种与癌症有关的疾病.本研究旨在诱导大肠杆菌RecQ解旋酶体外表达,并应用生物化学和生物物理学技术研究大肠杆菌RecQ解旋酶的生物学活性. 体外诱导表达获得纯度达90% 以上并具有高活性的大肠杆菌重组RecQ解旋酶,其可溶性好;经生物学活性分析显示具有DNA结合活性、ATP依赖的DNA解链活性、DNA依赖的ATP酶活性. 较之双链DNA（dsDNA）,大肠杆菌RecQ解旋酶更容易与单链DNA(ssDNA)结合( P<0.01 ),但与长度不同的dsDNA的结合特性有差异(P<0.01)而与ssDNA没有差异(P>0.05);大肠杆菌RecQ解旋酶对3种dsDNA的解链速率不同(P<0.05);大肠杆菌RecQ解旋酶的ATP酶活性与辅助因子ssDNA长度也呈正相关(P<0.01). 这些研究结果将有助于阐明大肠杆菌RecQ解旋酶的分子作用机制,并为研究RecQ解旋酶家族其它成员的结构与功能提供帮助.     

Human RECQ5beta, a protein with DNA helicase and strand-annealing activities in a single polypeptide

Proteins belonging to the highly conserved RecQ helicase family are essential for the maintenance of genomic stability. Here, we describe the biochemical properties of the human RECQ5beta protein. Like BLM and WRN, RECQ5beta is an ATP-dependent 3'-5' DNA helicase that can promote migration of Holliday junctions. However, RECQ5beta required the single-stranded DNA-binding protein RPA in order to mediate the efficient unwinding of oligonucleotide-based substrates. Surprisingly, we found that RECQ5beta possesses an intrinsic DNA strand-annealing activity that is inhibited by RPA. Analysis of deletion variants of RECQ5beta revealed that the DNA helicase activity resides in the conserved N-terminal portion of the protein, whereas strand annealing is mediated by the unique C-terminal domain. Moreover, the strand-annealing activity of RECQ5beta was strongly inhibited by ATPgammaS, a poorly hydrolyzable analog of ATP. This effect was alleviated by mutations in the ATP-binding motif of RECQ5beta, indicating that the ATP-bound form of the protein cannot promote strand annealing. This is the first demonstration of a DNA helicase with an intrinsic DNA strand-annealing function residing in a separate domain.     

A novel human hexameric DNA helicase: expression, purification and characterization

We have cloned, expressed and purified a hexameric human DNA helicase (hHcsA) from HeLa cells. Sequence analysis demonstrated that the hHcsA has strong sequence homology with DNA helicase genes from Saccharomyces cerevisiae and Caenorhabditis elegans, indicating that this gene appears to be well conserved from yeast to human. The hHcsA gene was cloned and expressed in Escherichia coli and purified to homogeneity. The expressed protein had a subunit molecular mass of 116 kDa and analysis of its native molecular mass by size exclusion chromatography suggested that hHcsA is a hexameric protein. The hHcsA protein had a strong DNA-dependent ATPase activity that was stimulated ≥5-fold by single-stranded DNA (ssDNA). Human hHcsA unwinds duplex DNA and analysis of the polarity of translocation demonstrated that the polarity of DNA unwinding was in a 5′→3′ direction. The helicase activity was stimulated by human and yeast replication protein A, but not significantly by E.coli ssDNA-binding protein. We have analyzed expression levels of the hHcsA gene in HeLa cells during various phases of the cell cycle using in situ hybridization analysis. Our results indicated that the expression of the hHcsA gene, as evidenced from the mRNA levels, is cell cycle-dependent. The maximal level of hHcsA expression was observed in late G₁/early S phase, suggesting a possible role for this protein during S phase and in DNA synthesis.     

DNA translocation activity of the multifunctional replication protein ORF904 from the archaeal plasmid pRN1

The replication protein ORF904 from the plasmid pRN1 is a multifunctional enzyme with ATPase-, primase- and DNA polymerase activity. Sequence analysis suggests the presence of at least two conserved domains: an N-terminal prim/pol domain with primase and DNA polymerase activities and a C-terminal superfamily 3 helicase domain with a strong double-stranded DNA dependant ATPase activity. The exact molecular function of the helicase domain in the process of plasmid replication remains unclear. Potentially this motor protein is involved in duplex remodelling and/or origin opening at the plasmid replication origin. In support of this we found that the monomeric replication protein ORF904 forms a hexameric ring in the presence of DNA. It is able to translocate along single-stranded DNA in 3′–5′ direction as well as on double-stranded DNA. Critical residues important for ATPase activity and DNA translocation activity were identified and are in agreement with a homology model of the helicase domain. In addition we propose that a winged helix DNA-binding domain at the C-terminus of the helicase domain could assist the binding of the replication protein specifically to the replication origin.     

A DNA helicase from Pisum sativum is homologous to translation initiation factor and stimulates topoisomerase I activity

DNA helicases play an essential role in all aspects of nucleic acid metabolism, by providing a duplex-unwinding function. This is the first report of the isolation of a cDNA (1.6 kb) clone encoding functional DNA helicase from a plant (pea, Pisum sativum). The deduced amino-acid sequence has eight conserved helicase motifs of the DEAD-box protein family. It is a unique member of this family, containing DESD and SRT motifs instead of DEAD/H and SAT. The encoded 45.5 kDa protein has been overexpressed in bacteria and purified to homogeneity. The purified protein contains ATP-dependent DNA and RNA helicase, DNA-dependent ATPase, and ATP-binding activities. The protein sequence contains striking homology with eIF-4A, which has not so far been reported as DNA helicase. The antibodies against pea helicase inhibit in vitro translation. The gene is expressed as 1.6 kb mRNA in different organs of pea. The enzyme is localized in the nucleus and cytosol, and unwinds DNA in the 3' to 5' direction. The pea helicase interacts with pea topoisomerase I protein and stimulates its activity. These results suggest that pea DNA helicase could be an important multifunctional protein involved in protein synthesis, maintaining the basic activities of the cell, and in upregulation of topoisomerase I activity. The discovery of such a protein with intrinsic multiple activity should make an important contribution to our better understanding of DNA and RNA transactions in plants.