C-terminal domain of the Uup ATP-binding cassette ATPase is an essential folding domain that binds to DNA

The Uup protein belongs to a subfamily of soluble ATP-binding cassette (ABC) ATPases that have been implicated in several processes different from transmembrane transport of molecules, such as transposon precise excision. We have demonstrated previously that Escherichia coli Uup is able to bind DNA. DNA binding capacity is lowered in a truncated Uup protein lacking its C-terminal domain (CTD), suggesting a contribution of CTD to DNA binding. In the present study, we characterize the role of CTD in the function of Uup, on its overall stability and in DNA binding. To this end, we expressed and purified isolated CTD and we investigated the structural and functional role of this domain. The results underline that CTD is essential for the function of Uup, is stable and able to fold up autonomously. We compared the DNA binding activities of three versions of the protein (Uup, UupΔCTD and CTD) by an electrophoretic mobility shift assay. CTD is able to bind DNA although less efficiently than intact Uup and UupΔCTD. These observations suggest that CTD is an essential domain that contributes directly to the DNA binding ability of Uup.     

SARS病毒受体ACE2的克隆、原核表达及其功能区鉴定

ACE2(angiotensin-converting enzyme 2,ACE2)是SARS冠状病毒(severe acute respiratory syndrome associatedcoronavirus,SARS-CoV)的主要受体。此研究旨在鉴定ACE2的SARS-CoV受体功能区,为进一步阐明SARS-CoV与细胞间的相互作用机制及研制抗病毒药物等提供理论依据。利用RT-PCR从Vero-E6细胞的mRNA中分两段扩增ACE2基因,其中N端片段ACE2A1-367(102~1 210nt)不包括ACE2的酶活性位点(1 223~1 237nt,或374~378aa),而C端片段ACE2B335-805(1 101~2 524nt)包括ACE2的酶活性位点。扩增片段克隆入pMD-18T,并进行测序鉴定。进一步构建与GST基因融合表达的原核表达质粒pGEX-ACE2A与pGEX-ACE2B,IPTG诱导表达。表达的融合蛋白分子量为65kD和77kD,主要以包涵体形式存在。Western blot证实表达产物具有免疫学活性。将纯化的包涵体蛋白质复性后进行Western blot分析,证实pGEX-ACE2A表达的蛋白(~65kD)能与SARS-CoV S1蛋白特异结合,而pGEX-ACE2B表达的蛋白(~77kD)不能与S1蛋白结合。结果表明,ACE2的受体活性与酶活性位点无关,受体功能区在ACE2 N端367个氨基酸内。     

蜜环菌糖苷水解酶家族基因在菌索与菌丝间的差异表达分析

蜜环菌是一种兼性腐生和寄生的真菌,通过降解伴栽基质并为药用植物(天麻)或菌物(猪苓)提供营养物质,而糖苷水解酶是这一过程的主要酶类.本研究从蜜环菌Armillaria mellea 541菌株转录数据库中共获得糖苷水解酶家族基因170个,分布于39个亚家族.进一步分析发现,这些家族基因编码的糖苷水解酶家族蛋白(glyc...     

正常小鼠高频心电图时域值和功率谱的研究

本文用南京新博公司生产的ＮＨＥ－１０００型心电高频信息检测分析仪研究了正常小鼠（昆明种）高频心电图（ＨＦ－ＥＣＧ）的时域值和ＱＲＳ波群的功率谱。主要结果如下（以正导为例,－Ｘ±ＳＤ）：心率６０３±８８次／ｍｉｎ（ｎ＝７４）;Ｐ－Ｒ间期相对较长。为３４．９±４．７ｍｓ（ｎ＝５８）,占心动周期的３４．９±４．９％,这与人类有很大的不同;ＱＲＳ波宽９．２±１．２ｍｓ,占心动周期的９．２±１．４％（ｎ＝７４）,这一结果与以前的文献报道相差较大。Ｔ波宽１０．３±３．２ｍｓ,占心动周期的１０．３±３．２％;Ｑ－Ｔ间期１９．４±３．２ｍｓ,占心动周期的１９．５±３．６％;ＱＲＳ波群峰－峰值（Ｖｐ－ｐ）为１．４５６±０．４８０ｍＶ;Ｔ波高０．３３６±０．１１５ｍＶ;７３只动物Ⅱ导联高频切迹总数只有３个,扭挫２６个。Ⅱ导联ＱＲＳ波群的功率谱特点：０—８０Ｈｚ的相对能量为４５．４８±１５．３２％;８０—２００Ｈｚ为４３．９７±９．９５％;２００—３００Ｈｚ为８．８９±７．３８％;３００—１０００Ｈｚ为１．６６±２．７４％。     

SorCS2 Controls Functional Expression of Amino Acid Transporter EAAT3 and Protects Neurons from Oxidative Stress and Epilepsy-Induced Pathology

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Protein Engineering Allows for Mild Affinity-based Elution of Therapeutic Antibodies

Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, Z_Ca, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the Z_Ca–Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification.     

Impact of single-chain Fv antibody fragment affinity on nanoparticle targeting of epidermal growth factor receptor-expressing tumor cells

To determine the importance of single-chain Fv (scFv) affinity on binding, uptake, and cytotoxicity of tumor-targeting nanoparticles, the affinity of the epidermal growth factor receptor (EGFR) scFv antibody C10 was increased using molecular evolution and yeast display. A library containing scFv mutants was created by error-prone PCR, displayed on the surface of yeast, and higher affinity clones selected by fluorescence activated cell sorting. Ten mutant scFv were identified that had a 3-18-fold improvement in affinity (KD=15-88 nM) for EGFR-expressing A431 tumor cells compared to C10 scFv (KD=264 nM). By combining mutations, higher affinity scFv were generated with KD ranging from 0.9 nM to 10 nM. The highest affinity scFv had a 280-fold higher affinity compared to that of the parental C10 scFv. Immunoliposome nanoparticles (ILs) were prepared using EGFR scFv with a 280-fold range of affinities, and their binding and uptake into EGFR-expressing tumor cells was quantified. At scFv densities greater than 148 scFv/IL, there was no effect of scFv affinity on IL binding and uptake into tumor cells, or on cytotoxicity. At lower scFv densities, there was less uptake and binding for ILs constructed from the very low affinity C10 scFv. The results show the importance of antibody fragment density on nanoparticle uptake, and suggest that engineering ultrahigh affinity scFv may be unnecessary for optimal nanoparticle targeting.     

从噬菌体展示随机12肽库中筛选与IRS-1PH结构域相结合的多肽模体

将胰岛素受体底物-1(IRS-1)的PH结构域(pleckstrin homology domain)编码序列克隆到融合表达载体pRSETA中,阳性克隆经IPTG诱导,表达出氨基端带6个连续组氨酸残基的融合蛋白。经检测,表达的目的蛋白一部分以可溶形式存在。利用Ni-NTA金属螯合亲和层析法在非变性条件下对表达的目的蛋白进行纯化,纯度大于98％。将纯化的目的蛋白包被于聚苯乙烯平皿上作为靶蛋白,经过4轮淘筛,得到能够与IRS-1的PH结构域相结合的重组噬菌体克隆;从中随机挑选出50个克隆进行DNA序列测定,对获得的短肽序列进行了分析。并通过ELISA检测了这些克隆与IRS-1的PH结构域的结合活性。     

茎瘤固氮根瘤菌ORS571 GGDEF和EAL结构域蛋白的预测及功能研究

[目的]环二鸟苷酸c-di-GMP是细菌中广泛存在的第二信使,能够调控多种细胞功能。c-di-GMP的合成与水解分别由含有GGDEF结构域和EAL结构域的蛋白催化。本研究针对茎瘤固氮根瘤菌ORS571的GGDEF和EAL结构域相关蛋白进行基因组学分析,并对三个同时含有GGDEF和EAL结构域的蛋白（AZC_3085、AZC_3226和AZC_4658）进行功能研究。[方法]利用SMART数据库对含有GGDEF和EAL结构域的蛋白进行结构域预测。利用CLUSTALW程序对蛋白序列进行比较分析。通过同源重组的方法构建突变株,并对突变株的细胞运动能力、胞外多糖合成、生物膜形成及与豆科宿主的结瘤等表型进行测定。[结果]茎瘤固氮根瘤菌ORS571中一共存在37个GGDEF和EAL结构域蛋白。突变株△4658的运动能力较野生型有下降,但是其胞外多糖合成能力、生物膜形成能力和竞争性结瘤能力较野生型有提高。此外,实验结果表明突变株△4658的胞内c-di-GMP水平高于野生型。突变株△3085和△3226的各种表型与野生型相比没有明显差异。[结论]茎瘤固氮根瘤菌ORS571编码如此大数量的GGDEF和EAL结构域蛋白,表明c-di-GMP可能在其信号转导过程中起到非常重要的作用。同时具有GGDEF和EAL结构域的蛋白AZC_4658对茎瘤固氮根瘤菌ORS571的运动能力、胞外多糖合成、生物膜形成及与宿主的结瘤起到一定的调节作用。