SSB蛋白与ssDNA相互作用的动力学和可视化研究

研究大肠杆菌单链结合蛋白(single-stranded DNA-binding protein,SSB)与单链DNA(single-stranded DNA,ssDNA)的相互作用对于了解其在DNA复制、重组和修复中的作用是非常重要的。通过表面等离子共振技术(surface plasmon resonance,SPR)得到了在有、无镁离子的情况下,SSB与ssDNA两者的平衡解离常数(equilibrium dissociation constant,KD)分别为9.67×10-7M和4.79×10-7M,阐明了镁离子对于两者作用形式的影响。利用原子力显微镜技术分别观察SSB蛋白、ssDNA和SSB-ssDNA复合物的成像,为下一步研究SSB在DNA代谢中作用模式的单分子可视化奠定了基础。     

逆转录病毒表达系统及其在外源蛋白高效表达中的应用

逆转录病毒表达系统是一种新的重组蛋白高效表达系统,它由逆转录病毒载体,包膜蛋白载体和包装细胞系构成,在基因治疗和生物制药领域都有很大的应用潜力。逆转录病毒和宿主细胞基因组的重组倾向于发生在转录活性区;口炎疱疹病毒-G蛋白 (vesicular stomatitis virus G, VSV-G)能有效扩大逆转录病毒感染宿主细胞的范围、提高逆转录病毒的感染效率;用高滴度的重组病毒感染细胞,经过简单的筛选即可获得高表达细胞株。本文对逆转录病毒表达系统的组成、感染的特点和机制及其应用前景作了概述。     

P.pastoris高效表达外源蛋白的研究进展

Ｐｉｃｈｉａ ｐａｓｔｏｒｉｓ表达系统具有原核细胞和哺乳类细胞表达系统特点,已广地应用于表达外源基因。为获得高质量外泊蛋白的表达而进行了大量研究并取得了许多进展,如构建并筛选多拷贝的由ＡＯＸ启动的表达盒,优化培液组分,减少蛋白降低,分泌蛋白的纯化和Ｎ－连接寡糖链的特点。     

淀粉样前体蛋白和LRP6在大肠杆菌中的表达及其体外相互作用

 淀粉样前体蛋白 (APP)是阿尔茨海默氏病 (AD)发病过程中有重要作用的蛋白 .利用酵母双杂交的方法发现低密度脂蛋白受体相关蛋白 6(LRP6)羧基端可和 APP羧基端片段相互作用 .分别构建了 APP和 LRP6的原核表达载体 ,并利用大肠杆菌获得 GST- APP1 0 6、MBP- LRP6融合蛋白 .体外相互作用研究证实了 APP羧基端和 LRP6羧基端之间的结合 .这使与 AD相关的两个重要蛋白 apo E和 APP联系起来 ,并提示 LRP6可能在 APP代谢和 Aβ产生中起重要作用 .     

金属硫蛋白突变体的植物高效表达载体的构建及其在烟草中的表达

金属硫蛋白有α、β两个结构域（ｄｏｍ ａｉｎ）,其中α结构域优先结合Ｃｄ２＋ 和Ｈｇ２＋ ．小鼠αα突变体在大肠杆菌中已经构建并得到表达,其转基因植株已得到,可在Ｃｄ３００（３００ μｍ ｏｌ／Ｌ）中生长．为了进一步提高外源基因在烟草中的表达量,首先用ＰＣＲ 的方法设计引物,在基因翻译起始密码子ＡＴＧ 附近加入植物偏爱的碱基组合ＡＡＣＡＡＴＧ．另外,将该突变体基因插入具有双３５ Ｓ（ＣａＭＶ３５Ｓ）强启动子的植物双元表达载体ｐＧＰＴＶｄ３５Ｓ－ＢＡＲ中,获得了带有αα突变体的植物双元表达载体．通过农杆菌介导的叶盘转化法转化烟草ＮＣ８９,获得了抗除草剂的转基因植株．经ＰＣＲ－Ｓｏｕｔｈｅｒｎ 和蛋白Ｄｏｔ－ｂｌｏｔｔｉｎｇ 检测,证明了αα突变体在烟草中的嵌合与表达．抗重金属实验证明转基因烟草可以在Ｃｄ４００（４００ μｍ ｏｌ／Ｌ）中生长．     

人造血相关PBX相互作用蛋白的原核表达及纯化鉴定

目的:构建带His标签的人造血相关PBX相互作用蛋白(HPIP)的原核表达载体,获得His-HPIP融合蛋白,并对其生物学功能进行初步检测。方法:以本实验室保存的pcDNA3.0-HPIP质粒为模板,采用PCR技术扩增HPIP编码序列,将其插入载体p ET-28a(+)中,经Bam HⅠ和HindⅢ双酶切鉴定后转化大肠杆菌Rossate株进行小量诱导,挑选能诱导出His-HPIP的菌液进行融合蛋白的纯化,采用SDS-PAGE和Western印迹检测融合蛋白的纯化效果,采用GST pull-down技术对蛋白的生物学功能进行初步鉴定。结果:双酶切和测序结果表明His-HPIP原核表达质粒构建成功;His pull-down实验证实His-HPIP蛋白和雌激素受体α存在相互作用,说明生物学活性良好。结论:原核表达并纯化出His-HPIP融合蛋白,为进一步研究HPIP在肿瘤发生发展中的功能奠定了基础。     

带myc标签的人造血相关PBX相互作用蛋白真核表达载体的构建及其功能研究

目的:构建带myc标签的造血相关PBX相互作用蛋白(HPIP)的真核表达载体,获得myc-HPIP融合蛋白,并对其生物学功能进行初步检测。方法:以本实验室保存的pcDNA3.0-HPIP质粒为模板,采用PCR技术扩增HPIP编码序列,将其插入pXJ-40-myc载体,通过Western印迹检测表达情况;将重组质粒与空载体分别转染人肝癌HepG2细胞,通过Western印迹检测其对AKT、ERK信号通路中AKT、ERK磷酸化水平的影响。结果:双酶切和测序结果表明myc-HPIP真核表达质粒构建成功,转染HepG2细胞后表达了myc-HPIP融合蛋白;Western印迹证明myc-HPIP可升高AKT、ERK的磷酸化水平。结论:构建了带myc标签的人HPIP真核表达载体,为进一步研究HPIP在肿瘤发生发展中的功能奠定了基础。     

P.pastoris高效表达外源蛋白的研究进展

Ｐｉｃｈｉａｐａｓｔｏｒｉｓ表达系统具有原核细胞和哺乳类细胞表达系统的特点,已广泛地应用于表达外源基因。为获得高质量外源蛋白的表达而进行了大量研究并取得了许多进展,如构建并筛选多拷贝的由ＡＯＸ启动的表达盒,优化培液组分,减少蛋白降解,分泌蛋白的纯化和Ｎ－连接寡糖链的特点。     

细胞膜蛋白与细胞骨架蛋白相互作用研究进展

细胞膜蛋白与胞浆骨架蛋白的相互作用对于维持细胞正常形态,细胞粘附与信号传导有重要作用,含有4.1/JEF结构域的蛋白4.1超家族与含有PDZ结构域的MAGUK蛋白家族能结合多种膜蛋白胞内区与胞浆蛋白,在膜蛋白与胞浆蛋白之间建立联系,对于细胞、细胞-细胞间连接的正常结构与功能的维持有着重要作用。     

外源蛋白在中国仓鼠卵巢细胞中高效表达的策略

高效表达外源蛋白,在生物制药中有重要意义.中国仓鼠卵巢细胞（Chinese hamster ovary cell）是表达外源蛋白的最佳真核表达系统之一.影响外源蛋白在CHO细胞表达的因素甚多,主要包括载体、宿主细胞和外源基因几方面.深入了解和灵活运用它们之间的关系,有助于获得外源基因在CHO细胞中的高效表达.     

Single-Stranded DNA-Binding Protein Complex from Helicobacter pylori Suggests an ssDNA-Binding Surface

Single-stranded DNA (ssDNA)-binding protein (SSB) plays an important role in DNA replication, recombination, and repair. SSB consists of an N-terminal ssDNA-binding domain with an oligonucleotide/oligosaccharide binding fold and a flexible C-terminal tail involved in protein-protein interactions. SSB from Helicobacter pylori (HpSSB) was isolated, and the ssDNA-binding characteristics of HpSSB were analyzed by fluorescence titration and electrophoretic mobility shift assay. Tryptophan fluorescence quenching was measured as 61%, and the calculated cooperative affinity was 5.4 × 10⁷ M^− 1 with an ssDNA-binding length of 25-30 nt. The crystal structure of the C-terminally truncated protein (HpSSBc) in complex with 35-mer ssDNA [HpSSBc-(dT)₃₅] was determined at a resolution of 2.3 Å. The HpSSBc monomer folds as an oligonucleotide/oligosaccharide binding fold with a Y-shaped conformation. The ssDNA wrapped around the HpSSBc tetramer through a continuous binding path comprising five essential aromatic residues and a positively charged surface formed by numerous basic residues.     

Orchestration of Haemophilus influenzae RecJ Exonuclease by Interaction with Single-Stranded DNA-Binding Protein

RecJ exonuclease plays crucial roles in several DNA repair and recombination pathways, and its ubiquity in bacterial species points to its ancient origin and vital cellular function. RecJ exonuclease from Haemophilus influenzae is a 575-amino-acid protein that harbors the characteristic motifs conserved among RecJ homologs. The purified protein exhibits a processive 5′-3′ single-stranded-DNA-specific exonuclease activity. The exonuclease activity of H. influenzae RecJ (HiRecJ) was supported by Mg^2 + or Mn^2 + and inhibited by Cd^2 +, suggesting a different mode of metal binding in HiRecJ as compared to Escherichia coli RecJ (EcoRecJ). Site-directed mutagenesis of highly conserved residues in HiRecJ abolished enzymatic activity. Interestingly, substitution of alanine for aspartate 77 resulted in a catalytically inactive enzyme that bound to DNA with a significantly higher affinity as compared to the wild-type enzyme. Noticeably, steady-state kinetic studies showed that H. influenzae single-stranded DNA-binding protein (HiSSB) increased the affinity of HiRecJ for single-stranded DNA and stimulated its exonuclease activity. HiSSB, whose C-terminal tail had been deleted, failed to enhance RecJ exonuclease activity. More importantly, HiRecJ was found to directly associate with its cognate single-stranded DNA-binding protein (SSB), as demonstrated by various in vitro assays. Interaction studies carried out with the truncated variants of HiRecJ and HiSSB revealed that the two proteins interact via the C-terminus of SSB protein and the core-catalytic domain of RecJ. Taken together, these results emphasize direct interaction between RecJ and SSB, which confers functional cooperativity to these two proteins. In addition, these results implicate SSB as being involved in the recruitment of RecJ to DNA and provide insights into the interplay between these proteins in repair and recombination pathways.     

Crystal Structure of an Unusual Single-Stranded DNA-Binding Protein Encoded by Staphylococcal Cassette Chromosome Elements

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人视黄醇结合蛋白在大肠杆菌中的高效表达及其活性测定

视黄醇结合蛋白 (retinol- binding protein,RBP)是体内结合、转运维生素 A的重要载体蛋白 ,对生长、繁殖、视觉及维持上皮细胞分化状态至关重要 ,不但是临床营养支持蛋白质营养评价的最灵敏指标 ,而且可作为慢性肾病、严重肝病、甲亢等相关疾病的辅助诊断指标 .同时 ,RBP可作为疏水小分子结合蛋白家族构效关系研究的模型 ,具有重要的理论与应用研究价值 .我们已克隆出了它的 c D-NA[1 ] ,本文报道了人 RBP在大肠杆菌中的高效表达及其产物的初步纯化和活性测定 .1　材料与方法1 .1　材料含目的片段的克隆质粒 p GEM- RBP为本室构建 …     

DEK蛋白的真核表达纯化及其活性检测

利用Bac-to-Bac杆状病毒表达系统表达DEK蛋白并进行纯化。首先以pFastBacI质粒构建重组质粒pFastBacI-DEK,转化DH10Bac大肠杆菌后获得重组穿梭载体Bacmid-DEK,通过脂质体介导转染Sf9细胞产生具有强感染力的重组杆状病毒AcNPV-DEK。用此重组杆状病毒AcNPV-DEK感染Sf9细胞表达His-DEK融合蛋白。在非变性条件下,利用Ni-NTA agarose对表达的His-DEK融合蛋白进行纯化,经SDS-PAGE和Western blotting分析,在50 kDa处出现特异性蛋白条带并证实其为His-DEK融合蛋白。凝胶迁移阻滞实验表明,融合蛋白His-DEK与DNA 的结合具有结构特异性,其与超螺旋型DNA结合活性强于与线性化DNA的结合活性。真核表达并纯化的融合蛋白His-DEK与DNA的结合活性要明显强于原核表达的融合蛋白His-CDB。DEK 蛋白的磷酸化修饰会阻碍其与DNA的结合,而Sf9细胞中表达的融合蛋白His-DEK存在磷酸化修饰,将His-DEK去磷酸化后,其与DNA的结合活性有所提高。     

Identification and Characterization of the Single-Stranded DNA-Binding Protein of Bacteriophage P1

The genome of bacteriophage P1 harbors a gene coding for a 162-amino-acid protein which shows 66% amino acid sequence identity to the Escherichia coli single-stranded DNA-binding protein (SSB). The expression of the P1 gene is tightly regulated by P1 immunity proteins. It is completely repressed during lysogenic growth and only weakly expressed during lytic growth, as assayed by an ssb-P1/lacZ fusion construct. When cloned on an intermediate-copy-number plasmid, the P1 gene is able to suppress the temperature-sensitive defect of an E. coli ssb mutant, indicating that the two proteins are functionally interchangeable. Many bacteriophages and conjugative plasmids do not rely on the SSB protein provided by their host organism but code for their own SSB proteins. However, the close relationship between SSB-P1 and the SSB protein of the P1 host, E. coli, raises questions about the functional significance of the phage protein.     

A Phage Single-Stranded DNA (ssDNA) Binding Protein Complements ssDNA Accumulation of a Geminivirus and Interferes with Viral Movement

Geminiviruses are plant viruses with circular single-stranded DNA (ssDNA) genomes encapsidated in double icosahedral particles. Tomato leaf curl geminivirus (ToLCV) requires coat protein (CP) for the accumulation of ssDNA in protoplasts and in plants but not for systemic infection and symptom development in plants. In the absence of CP, infected protoplasts accumulate reduced levels of ssDNA and increased amounts of double-stranded DNA (dsDNA), compared to accumulation in the presence of wild-type virus. To determine whether the gene 5 protein (g5p), a ssDNA binding protein from Escherichia coli phage M13, could restore the accumulation of ssDNA, ToLCV that lacked the CP gene was modified to express g5p or g5p fused to the N-terminal 66 amino acids of CP (CP66:6G:g5). The modified viruses led to the accumulation of wild-type levels of ssDNA and high levels of dsDNA. The accumulation of ssDNA was apparently due to stable binding of g5p to viral ssDNA. The high levels of dsDNA accumulation during infections with the modified viruses suggested a direct role for CP in viral DNA replication. ToLCV that produced the CP66:6G:g5 protein did not spread efficiently in Nicotiana benthamiana plants, and inoculated plants developed only very mild symptoms. In infected protoplasts, the CP66:6G:g5 protein was immunolocalized to nuclei. We propose that the fusion protein interferes with the function of the BV1 movement protein and thereby prevents spread of the infection.     

克隆、表达及纯化核糖体蛋白S6用于体外激酶活性检测

目的:构建40S核糖体蛋白S6的原核表达载体,表达并纯化S6蛋白,将其作为底物用于S6激酶(S6K)的体外活性测定。方法:采用RT-PCR方法从人胚肾细胞HEK293中获取S6 cDNA,将扩增产物克隆至大肠杆菌表达载体中,进行酶切及测序鉴定;IPTG诱导GST-S6融合蛋白在大肠杆菌中表达,用谷胱甘肽亲和层析纯化GST-S6,免疫沉淀法检测该蛋白是否可作为底物用于S6K的体外激酶活性测定。结果:酶切及测序鉴定表明构建了S6原核表达载体,并表达及纯化出GST-S6融合蛋白,相对分子质量为55×103。该蛋白可用于S6K的体外激酶活性测定,特异性强。结论:S6蛋白的克隆、表达与纯化成功,可用于S6K的体外激酶活性测定,为研究S6K的功能奠定了基础。