重组人CK2β亚基的原核表达、纯化与鉴定

将构建成功的人蛋白激酶CK2β亚基cDNA的重组质粒, 转化大肠杆菌BL21 (DE3), 在IPTG诱导下表达. 表达蛋白大多数以不溶形式存在. 6L(约10.2 g)表达菌抽提得到约20 mg的可溶性表达产物, 通过P11磷酸纤维素一步层析分离, 得到6.8 mg纯化蛋白. SDS-聚丙烯酰胺凝胶电泳结果显示纯化的蛋白为一分子质量26 ku的单一蛋白带.蛋白质印迹结果证明：纯化的表达产物与抗人CK2β抗体可发生特异性免疫反应. CK2β亚基对CK2α有激活作用, 纯化的CK2α和β亚基在等摩尔混合时即可组成有最大生物活性的全酶. 实验结果有力地证明了克隆表达与纯化的重组蛋白是人蛋白激酶CK2β亚基.     

人蛋白激酶CK2α′亚基在大肠杆菌中的克隆、表达及其重组蛋白的纯化与性质鉴定

通过RT PCR从HL 6 0细胞获得人蛋白激酶CK2α′亚基编码区cDNA ,将NdeⅠ HindⅢ双酶切的PCR产物和pT7 7表达载体进行定向克隆、细菌转化、电泳初筛和限制性酶切分析鉴定 .随机挑选阳性克隆进行DNA测序确证 ,筛选含与已知序列完全相符的重组质粒 (命名为pTCKA′) .将其转化BL2 1(DE3)菌 ,IPTG诱导后未见高效特异表达 .然后将人CK2α′cDNA亚克隆至GST融合蛋白表达载体 ,经同样转化和诱导步骤后可见一蛋白特异高效表达 .Western印迹结果证明 :该蛋白能与兔抗人CK2α′3 3 3 3 50 肽段抗血清发生特异性免疫反应 .采用GSH Sepharose 4B柱纯化 ,凝血酶酶切 ,最后从 4g细菌获 4 4mg纯化重组蛋白 .通过性质鉴定和酶动力学分析证明 :克隆、表达和纯化的重组蛋白是有生物学活性的人CK2α′亚基 .     

重组人蛋白激酶CK2β亚基cDNA的克隆与测序

蛋白激酶ＣＫ２是一种存在的信使非依赖性丝／苏氨酸蛋白激酶．它是由两个催化亚基（α或α′）和两个调节亚基（β）组成的不均一四聚体．用反转录ＰＣＲ从ＨＬ－６０细胞中获得了人蛋白激酶ＣＫ２β亚基编码区ｃＤＮＡ,将ＮｄｅⅠ／ＨｉｎｄⅢ双酶切ＰＣＲ产物连接到表达载体ｐＴ７－７的ＮｄｅⅠ／ＨｉｎｄⅢ双酶切位点中．转化感受态细菌ＤＨ５α获得转化子,阳性筛选率为７２％．限制性酶切分析结果表明,插入片段和重组质粒的大小与理论推测值相符．随机挑选４个阳性质粒测定其插入片段ＤＮＡ序列,结果显示有２个含有正确插入的人蛋白激酶ＣＫ２βｃＤＮＡ,命名为ｐＴＣＫＢ．其余２个克隆分别存在１个和２个点突变,即在其编码区ｃｏｎｄｏｎ１４８的ＴＣＡ→ＴＴＡ,结果Ｓｅｒ→Ｌｅｕ;另一个则在Ｃｏｎｄｏｎ１４３ＧＴＧ→ＡＴＧ,Ｖａｌ→Ｍｅｔ;Ｃｏｎｄｏｎ１７０ＧＴＧ→ＧＣＧ,Ｖａｌ→Ａｌａ．重组质粒（ｐＴＣＫＢ）克隆的成功,将为在原核细胞中表达蛋白激酶ＣＫ２β亚基以及利用ＣＫ２βｃＤＮＡ作为探针进行深入研究打下基础．并为利用ｐＴ７－７表达载体构建其他重组质粒建立了一套成功的方法     

重组人TNF-α在大肠杆菌表达、纯化及生物学活性鉴定

[目的]获得人TNF-α蛋白,并鉴定其生物学活性。[方法]通过对人源TNF-α序列进行密码子优化构建了p Cold-TNFα原核表达载体,完成了TNF-α重组蛋白的表达,建立了两步分离纯化方案,并利用Western Blot和细胞刺激实验检测了重组人TNF-α蛋白抗原性和生物学活性。[结果]利用大肠杆菌成功表达重组人TNF-α蛋白,经酶切纯化后的人TNF-α蛋白不含标签序列,纯度达95%,收率约70%,并具有良好的生物学活性。[结论]获得具有生物学活性的人TNF-α蛋白,为进一步的中和抗体研究奠定了基础。     

蛋白激酶CK2α在人喉癌细胞Hep-2中的表达及意义

目的研究蛋白激酶CK2α(Protein kinase CK2α,PK-CK2α)在人喉癌细胞Hep-2中的表达,探讨其与喉癌之间的关系。方法体外培养Hep-2细胞和Hacat细胞,应用半定量逆转录-聚合酶链反应(RT-PCR)技术分别检测蛋白激酶CK2αmRNA在Hep-2细胞和Hacat细胞中的表达;应用免疫细胞化学技术分别检测蛋白激酶CK2α蛋白在Hep-2细胞和Hacat细胞中的表达。结果蛋白激酶CK2αmRNA在Hep-2细胞中高表达,在Hacat细胞中低表达,差异有统计学意义(P<0.01)。蛋白激酶CK2α蛋白在Hep-2细胞中高表达,在Hacat细胞中低表达,差异有统计学意义(P<0.01)。结论蛋白激酶CK2α过表达可能与喉癌的发生和发展有关。     

CK2 β的蛋白表达与纯化及其RNA沉默的研究

蛋白激酶CK2是一个丝氨酸/苏氨酸特异性的激酶,通常以异源四聚体形式存在,由两个催化亚基(CK2α和CK2α′)和两个调节亚基CK2β组成。CK2激酶α亚基含有和其它蛋白激酶的催化功能域同源的激酶功能域。CK2激酶β亚基在调节a亚基的基本活性方面起着很复杂的作用。通过CK2β的结合活性,CK2α改变它的活性和作用底物。本研究成功的表达和纯化了CK2β蛋白,并且构建了CK2β的S iRNA载体,为其进一步功能研究做好准备。     

重组人α2b型干扰素的纯化与鉴定

采用单克隆抗体亲和层析一步纯化法对大肠杆菌表达的重组人a2b型干扰素(IFN－α2b)进行了纯化,然后利用凝胶过滤高压液相层析、SDS－聚丙烯酰胺凝胶电泳和N端25个氨基酸的序列测定等方法对纯化产品进行了鉴定,表明一步纯化的IFN—α2b达到95%以上的纯度,其比活性为2．54×10⁸U／mg但重组IFN—α2b产品N端不均一,含有约30％的去Met分子和约70％的带Met分子。从蛋白质序列的水平上证实,本实验室用定位诱变法构建的IFN—α2b基因在大肠杆菌系统中得到了正确的表达。     

拟南芥FT基因原核表达载体的构建、表达和蛋白纯化

FT基因是植物成花素,在植物的开花调控中起着重要作用。构建了用于原核表达的FT-eGFP表达载体,并在大肠杆菌BL21中进行重组蛋白的诱导表达。从IPTG诱导浓度、诱导时间和诱导温度等方面进行了细致的分析,最终建立了FT-eGFP融合蛋白诱导表达的优化体系:当菌液OD600=0.6-1.0时,采用IPTG1.0mol/L,在28℃诱导表达6h。摸索和建立了利用HisTrapKit标签,经过镍柱纯化,纯化目的蛋白的技术体系,为进一步研究FT基因在植物开花调控中的应用奠定了基础。     

重组人源脑红蛋白的高效表达、纯化及鉴定

脑红蛋白(Neuroglobin,NGB)是新发现的主要表达于脊椎动物神经元及视网膜中的第三类携氧珠蛋白。已有诸多报道认为脑红蛋白在缺氧缺血性脑损伤的神经保护过程中,作为活性氧簇(ROS)的清除剂或缺氧信号的感受器起重要作用,但其神经保护作用的具体机制不明。显然,实现该蛋白的制备对于研究其功能具有重要作用。基于此,通过RT-PCR从人胎脑中扩增出脑红蛋白cDNA并克隆到原核表达载体pBV220,通过测序鉴定正确后转化至大肠杆菌HB101中诱导表达,表达产物超声破碎后经凝胶过滤柱Sephacryl S-200和阴离子交换柱Q Sepharose FF纯化,最后通过Sephadex G-25脱盐。经15% SDS-PAGE和Western blot检测及质谱和蛋白序列分析鉴定制备的蛋白样品为脑红蛋白。本文采用两步法实现了脑红蛋白高效特异性纯化,为后期基于脑红蛋白神经保护作用的功能及机制研究奠定了重要基础。     

人源酪蛋白激酶Ⅱ催化亚基的构建及表达纯化

目的:构建人源CK2催化亚基pET-28a(+)-hcsnk2a1和pET-28a(+)-hcsnk2a2重组质粒,并进行原核表达纯化得到高纯度融合蛋白,为进一步开展CK2生理病理机制研究以及CK2作为肿瘤抑制靶点相关抑制剂的筛选和评价提供实验基础。方法:利用合成的人源csnk2a1和csnk2a2目的基因片段,将酶切连接得到的重组质粒经测序验证后,进行大肠杆菌感受态BL21 (DE3)/Transetta (DE3)转化。使用合适浓度IPTG诱导融合蛋白的表达以得到可溶性的融合蛋白,并应用AKTA avant蛋白纯化仪和Ni2+-NTA预装柱进行纯化,蛋白纯度最后经SDS-PAGE胶分离后考马斯亮蓝染色和Western Blot检测鉴定。结果:测序结果表明pET-28a(+)-hcsnk2a1和pET-28a(+)-hcsnk2a2质粒均成功被构建;经转化诱导表达后,成功纯化得到相对分子量为42KD的his-hcsnk2a1和38KD的his-hcsnk2a2目的融合蛋白。结论:首次成功构建得到pET-28a(+)-hcsnk2a1和pET-28a(+)-hcsnk2a2质粒;并表达纯化得到高浓度和高纯度的his-hcsnk2a1和his-hcsnk2a2融合蛋白,为后期的蛋白功能研究和抑制剂筛选评价提供了基础。     

Coordinate expression of the alpha and beta subunits of heterotrimeric G proteins involves regulation of protein degradation in CHO cells

Individual cell types express a characteristic balance between heterotrimeric G protein alpha and betagamma subunits, but little is known about the regulatory mechanism. We systemically examined the regulatory mechanism in CHO cells. We found that expression of Galphas, Galphai2, and Galphaq proteins increased in direct proportion to the increase of Gbeta1gamma2 overexpressed transiently. Expression of Gbeta protein also increased following overexpression of Galphas, Galphai2, and Galphaq. The Gbetagamma overexpression stimulated degradation of Gbeta in contrast to reduction of Galphas degradation. We conclude that coordinate expression of the G protein subunits involves regulation of protein degradation via proteasome in CHO cells.     

The multiple personalities of the regulatory subunit of protein kinase CK2: CK2 dependent and CK2 independent roles reveal a secret identity for CK2beta

Protein kinase CK2 (formerly casein kinase II), an enzyme that participates in a wide variety of cellular processes, has traditionally been classified as a stable tetrameric complex consisting of two catalytic CK2alpha or CK2alpha' subunits and two regulatory CK2beta subunits. While consideration of CK2 as a tetrameric complex remains relevant, significant evidence has emerged to challenge the view that its individual subunits exist exclusively within these complexes. This review will summarize biochemical and genetic evidence indicating that the regulatory CK2beta subunit exists and performs functions independently of CK2 tetramers. For example, unbalanced expression of catalytic and regulatory CK2 subunits has been observed in a variety of tissues and tumors. Furthermore, localization studies including live cell imaging have demonstrated that while the catalytic and regulatory subunits of CK2 exhibit extensive co-localization, independent mobility of the individual CK2 subunits can also be observed within cells. Identification of proteins that interact with CK2beta in the absence of catalytic CK2 subunits reinforces the notion that CK2beta has functions distinct from CK2 and begins to offer insights into these CK2-independent functions. In this respect, the discovery that CK2beta can interact with and modulate the activity of a number of other serine/threonine protein kinases including A-Raf, c-Mos and Chk1 is particularly striking. This review will discuss the interactions between CK2beta and these protein kinases with special emphasis on the properties of CK2beta that mediate these interactions and on the implications of these interactions in yielding new prospects for elucidation of the cellular functions of CK2beta.     

Highlighting protein kinase CK2 movement in living cells

Protein kinase CK2 has traditionally been described as a stable heterotetrameric complex (α < eqid1 > β₂) but new approaches that effectively capture the dynamic behavior of proteins, are bringing a new picture of this complex into focus. To track the spatio-temporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with GFP and analog proteins. Beside the mostly nuclear localization of both subunits, and the identification of specific domains on each subunit that triggers their localization, the most significant finding was that the association of both CK2 subunits in a stable tetrameric holoenzyme eliminates their nuclear import (Mol Cell Biol {23}: 975–987, 2003). Molecular movements of both subunits in the cytoplasm and in the nucleus were analyzed using different new and updated fluorescence imaging methods such as: fluorescence recovery after photo bleaching (FRAP), fluorescence loss in photo bleaching (FLIP), fluorescence correlation spectroscopy (FCS), and photoactivation using a biphoton microscope. These fluorescence-imaging techniques provide unprecedented ways to visualize and quantify the mobility of each individual CK2 subunit with high spatial and temporal resolution. Visualization of CK2 heterotetrameric complex formation could also be recorded using the fluorescence resonance energy transfer (FRET) technique. FRET imaging revealed that the assembling of this molecular complex can take place both in the cytoplasmic and nuclear compartments. The spatio–temporal organization of individual CK2 subunits and their dynamic behavior remain now to be correlated with the functioning of this kinase in the complex environment of the cell.     

Isolation of Human Serum Alpha-2 HS Glycoprotein

Alpha-2 HS glycoprotein was obtained in an almost pure state from normal human serum, by ion exchange chromatography followed by curtain electrophoresis, gel filtration and ammonium sulfate precipitation. Alpha-2 HS glycoprotein has a sedimentation coefficient of 3.5 S, and an approximate molecular weight of 49,000 Dal tons. Purified α₂HS glycoprotein enhances phagocytosis and may be the human homologue of rat α₂ opsonic protein.     

Evolved to be active: sulfate ions define substrate recognition sites of CK2alpha and emphasise its exceptional role within the CMGC family of eukaryotic protein kinases

CK2alpha is the catalytic subunit of protein kinase CK2 and a member of the CMGC family of eukaryotic protein kinases like the cyclin-dependent kinases, the MAP kinases and glycogen-synthase kinase 3. We present here a 1.6 A resolution crystal structure of a fully active C-terminal deletion mutant of human CK2alpha liganded by two sulfate ions, and we compare this structure systematically with representative structures of related CMGC kinases. The two sulfate anions occupy binding pockets at the activation segment and provide the structural basis of the acidic consensus sequence S/T-D/E-X-D/E that governs substrate recognition by CK2. The anion binding sites are conserved among those CMGC kinases. In most cases they are neutralized by phosphorylation of a neighbouring threonine or tyrosine side-chain, which triggers conformational changes for regulatory purposes. CK2alpha, however, lacks both phosphorylation sites at the activation segment and structural plasticity. Here the anion binding sites are functionally changed from regulation to substrate recognition. These findings underline the exceptional role of CK2alpha as a constitutively active enzyme within a family of strictly controlled protein kinases.     

肺炎克雷伯菌调控蛋白RcsAB的构建表达及活性鉴定

为了探讨转录调控子Rcs AB对靶基因的转录调控作用,构建肺炎克雷伯菌RcsA和RcsB的重组质粒,之后诱导蛋白表达,提取纯化后测定其活性。PCR得到rcsA、rcsB片段,分别将两DNA片段克隆至表达载体pMAL-C5X、pET28a,构建重组质粒。再将重组质粒导入E. coli BL21(DE3)菌株中,经IPTG诱导后收集菌体,超声破碎。破碎后的上清过柱、透析纯化,得到高纯度的蛋白,通过EMSA进行蛋白活性鉴定。RcsA,RcsB蛋白成功表达纯化,并能够与靶基因结合,初步证明蛋白具有生物学活性。成功制备有生物学活性的RcsA、RcsB蛋白,为进一步研究RcsAB蛋白复合物特异的生物学功能提供物质基础。