半乳糖凝集素-1的融合克隆及纯化

目的：表达和纯化半乳糖凝集素-1融合蛋白。方法：用PCR方法从乳腺文库中扩增半乳糖凝集素-1编码序列,将其以正确相位与pGEX-KG载体中的GST编码序列融合,将重组质粒转化大肠杆菌DH5α后,用谷胱甘肽-Sepharose 4B纯化融合蛋白,并用Western印迹检测融合蛋白的表达。结果：构建得到半乳糖凝集素-1的融合蛋白表达载体;Western印迹检测表明,GST-半乳糖凝集素-1融合蛋白成功表达,并纯化得到融合蛋白。结论：克隆和表达了半乳糖凝集素-1基因,并得到纯化的融合蛋白。     

OS-9基因的融合表达、纯化及多克隆抗体制备

OS 9基因广泛表达于人体多种组织 ,该基因的表达产物可能与肿瘤的发生相关 .为获得可溶性表达的OS 9蛋白 ,制备多克隆抗体 ,深入了解OS 9基因的功能 ,将OS 9基因片段克隆入组氨酸标签融合的表达载体pET2 8a中 ,IPTG诱导 ,利用金属螯合亲和层析 (MCAC)进行纯化 ,薄层扫描及Bradford法检测纯化蛋白的纯度与含量 .免疫家兔制备多克隆抗体 ,利用间接ELISA法检测抗体效价 ,Western印迹检测抗体特异性 .经表达形式分析证明 ,融合蛋白大部分可溶 .薄层扫描分析纯度可达 90 %以上 ,Bradford法检测蛋白浓度约 0 1mg ml.抗血清效价可达 1∶32 0 0以上 ,Western印迹检测证明抗体特异性良好 .经诱导表达及纯化制备出可溶的纯度较高的OS 9蛋白产物 ,并获得高效价特异性良好的多克隆抗体     

转录因子XBP1的融合表达、纯化及多克隆抗体的制备

人X盒结合蛋白 1(XBP 1)为一种转录因子 ,与多种肿瘤的发生、发展有密切关系 .XBP 1有2种剪切形式 ,即XBP 1S和XBP 1U .将这 2种剪切形式中的一段相同编码序列 (编码 82～ 14 7位氨基酸 )重组于谷胱甘肽S转移酶 (GST)融合蛋白表达载体pGEX KG中 ,构建成重组质粒pGST XBP 1(82～ 14 7位氨基酸 ) .将该重组质粒转化E .coliDH5α后 ,表达GST XBP 1(82～ 14 7位氨基酸 )融合蛋白 ,经谷胱甘肽 Sepharose 4B亲和层析获得纯化的融合蛋白 .用此融合蛋白免疫家兔制备多克隆抗体 .利用制备的抗体分别用Western印迹和免疫细胞化学检测XBP 1的 2种剪切形式在哺乳动物细胞中的表达 .结果表明 ,该抗体对XBP 1的 2种剪切形式均具有反应原性 ,效价高 ,特异性好 ,可以用于进一步研究XBP 1的功能     

人SUMO-3基因原核表达及多克隆抗体制备

构建人SUMO-3基因的原核表达载体pET41a(+)-SUMO-3,表达重组GST-SUMO-3融合蛋白,制备人SUMO-3多克隆抗体。试验结果显示,通过PCR方法从重组质粒pEYFP-SUMO-3中克隆到的SUMO-3 N端93个氨基酸的基因序列与NCBI上提供的序列一致,重组质粒pET41a(+)-SUMO-3构建成功;重组pET41a(+)-SUMO-3在E.coli.BL21 (DE3) pLysS中表达GST-SUMO-3融合蛋白,分子量为44.0 kDa,与预期分子量一致;采用亲和层析纯化融合蛋白GST-SUMO-3并免疫家兔,获得人SUMO-3抗体;Western blot 检测显示该抗体可以特异性识别SUMO-3,ELISA检测结果成阳性,抗体效价约为1: 20000。实验结果为进一步研究人SUMO-3及SUMO第二类家族的功能提供了有用工具。     

Memo蛋白的融合表达和纯化

目的:一些临床预后较差的恶性肿瘤一般具有很高的转移性和侵袭性,近来的研究表明,Memo蛋白与恶性肿瘤的这些特性有十分密切的关系。本研究拟克隆、融合表达和纯化Memo蛋白。方法:用PCR方法从乳腺文库中扩增Memo蛋白编码序列,并将其以正确相位与pGST载体中的GST编码序列融合,构建成重组质粒pGST-Memo。将此重组质粒转化大肠杆菌DH5α后,用谷胱甘肽-Sepharose4B纯化融合蛋白,并用Western印迹检测融合蛋白的表达。结果:构建了Memo蛋白的融合表达载体,并在大肠杆菌DH5α中得到表达,经谷胱甘肽-Sepharose4B亲和层析获得了纯化的GST-Memo融合蛋白。Western印迹检测表明,此蛋白可与GST抗体反应。结论:Memo蛋白的克隆、融合表达和纯化,为进一步研究恶性肿瘤的转移性、侵袭性及其相关机理提供了有用的材料。     

GST/MKRN1融合蛋白的表达、纯化及多克隆抗体制备

目的:利用大肠杆菌BL21(DE3)表达GST/MKRN1融合蛋白,亲和层析分离纯化目的蛋白,进行动物免疫制备多克隆抗体。方法:MKRN1cDNA全长1449bp,编码482个氨基酸残基。将其基因片段克隆到pGEX-4T-1载体上,获得pGEX-4T-1-MKRN1原核表达重组质粒,转化大肠杆菌BL21(DE3),经IPTG诱导表达,在大肠杆菌表达系统中获得可溶性表达,经谷胱甘肽Sepharose4B介质填充的层析柱分离纯化蛋白,制备抗原免疫动物,得到pGEX-4T-1-MKRN1多克隆抗体。结果:ELISA结果显示血清抗体效价可达到1∶256000。通过Western免疫印迹及免疫细胞荧光分析,自制的多克隆抗体能特异地与MKRN1蛋白相互作用,可用于免疫细胞化学分析。结论:制备了效价和特异性良好的抗MKRN1多克隆抗体,经实验验证获得的免疫血清能够满足针对MKRN1的Western免疫印迹和细胞免疫组化检测的实验要求,为今后深入研究MKRN1表达的组织分布、细胞内定位及与端粒酶催化亚基(hTERT)之间相互作用的生物学意义提供了有用的实验工具。     

核心组蛋白的融合表达和纯化

目的:克隆核心组蛋白H2A、H2B、H3和H4的基因,表达并纯化组蛋白与谷胱甘肽S-转移酶(GST)的融合蛋白。方法:用PCR方法从乳腺文库中扩增核心组蛋白H2A、H2B、H3和H4的编码序列,分别将其以正确相位与pGEX-KG载体中的GST编码序列融合,得到重组质粒pGST-H2A、pGST-H2B、pGST-H3和pGST-H4,分别转化大肠杆菌BL21,表达融合蛋白GST-H2A、GST-H2B、GST-H3和GST-H4;用谷胱甘肽-Sepharose 4B亲和纯化融合蛋白;用Western印迹检测融合蛋白的表达及纯化。结果:分别构建了核心组蛋白H2A、H2B、H3和H4的融合表达载体;Western印迹检测表明,融合蛋白GST-H2A、GST-H2B、GST-H3和GST-H4获得表达及纯化。结论:表达并纯化了H2A、H2B、H3和H4的融合蛋白,为进一步研究核心组蛋白的功能奠定了基础。     

FHL家族的克隆及其融合蛋白的纯化

目的:纯化FHL家族(FHL1～FHL5)融合表达蛋白。方法:用PCR方法从乳腺文库和卵巢文库中扩增FHL1～FHL5编码序列,将其以正确相位与pGEX-KG载体中的GST编码序列融合,将重组质粒分别转化E.coliDH5α后,用谷胱甘肽-Sepharose4B纯化融合蛋白,并用Westernblot检测融合蛋白的表达。结果:构建得到FHL1～FHL5的融合表达载体,West-ernblot检测表明GST-FHL1～GST-FHL5共5种融合蛋白均成功表达,并纯化得到融合蛋白。结论:成功克隆FHL1～FHL5基因,并得到纯化的融合蛋白。     

GST-SOD1-R9融合蛋白的表达、纯化、稳定性与跨膜效应

穿膜肽TAT介导的双效抗氧化酶GST(谷胱甘肽巯基转移酶)-TAT-SOD1(Cu,Zn超氧化物歧化酶),可有效清除胞内自由基,其预防氧化损伤的效果强于SOD1-TAT,但前者的跨膜能力不如后者。为增强双效抗氧化酶的跨膜效率,本研究融合了SOD1和穿膜肽R9,合成SOD1-R9全基因序列,并将其插入带有GST的原核表达载体pGEX-4T-1中,成功构建了GST-SOD1-R9融合蛋白表达质粒。然后,将重组质粒pGEX-4T-1-SOD1-R9转化大肠杆菌BL21(DE3),用IPTG诱导表达融合蛋白,通过改变诱导温度和诱导时间,确定了融合蛋白在25℃下表达11 h,可得到高表达量的可溶性GST-SOD1-R9融合蛋白。利用80%硫酸铵沉淀和GST琼脂糖树脂纯化得到纯蛋白,应用SDS-PAGE和酶活性鉴定纯化的蛋白为正确表达的目标蛋白。GST-SOD1-R9融合蛋白的温度和pH稳定性实验结果证实,该蛋白在生理条件下具有良好的SOD和GST活性。细胞跨膜实验结果证明其跨膜能力与GST-TAT-SOD1融合蛋白相比显著增强(P0.05)。这些工作为深入研究GST-SOD1-R9的抗氧化损伤效应建立了基础。     

类泛素化修饰蛋白SUMO1的表达纯化及抗体制备

SUMO是近年发现的类泛素化修饰蛋白,可通过异肽键共价连接到靶蛋白上,影响靶蛋白的细胞内定位、稳定性及与其它生物大分子的相互作用. 为研究蛋白质的SUMO化修饰,本文表达并利用亲和层析的方法纯化了重组的人SUMO1,制备了兔抗hSUMO1的多克隆抗体. 经ELISA和免疫印迹检测,获得了灵敏度高、特异性好的抗体,可用于SUMO化修饰靶蛋白的鉴定及SUMO化修饰的生物学功能研究.     

Caspase recruitment domain of procaspase-2 could be a target for SUMO-1 modification through Ubc9

To identify the binding proteins that regulate the function of procaspase-2, we screened for proteins using the yeast two-hybrid method and isolated human Ubc9 and SUMO-1 as the candidates. Ubc9 and SUMO-1 interacted with the caspase recruitment domain of procaspase-2 in its N-terminal. We elucidated the covalent modification of procaspase-2 by SUMO-1 in mammalian cells by immunoprecipitation followed by Western blot analysis. Procaspase-2 and SUMO-1 were co-localized by dot-like structures in the nucleus that are related to promyelocytic leukemia bodies. Interestingly, a conjugation-deficient mutant (K60R) procaspase-2 resulted in a delay of its enzyme maturation (appearance of p12 subunit) compared to that of wild-type. Thus, the modification with SUMO-1 may play a critical role in the nuclear localization and the activation (maturation) of procaspase-2.     

A Kruppel zinc finger of ZNF 146 interacts with the SUMO-1 conjugating enzyme UBC9 and is sumoylated in vivo

The OZF (ZNF146) protein is a 33 kDa Kruppel protein, composed solely of 10 zinc finger motifs. It is overexpressed in the majority of pancreatic cancers and in more than 80% of colorectal cancers. We have identified OZF interacting factors with a yeast two-hybrid screen. Half of the positive clones characterized encoded UBC9, the E2 enzyme involved in the covalent conjugation of the small ubiquitin-like modifier 1 (SUMO-1). SUMO-1 is a 17 kDa migrating protein that is conjugated to several proteins and has been reported to exhibit multiple effects, including modulation of protein stability, subcellular localization, and gene expression. In HeLa cells transfected with OZF and SUMO-1 expression vectors, immunoblot revealed a major band migrating at 50 kDa and a minor band at 67 kDa, corresponding to the attachment to OZF of one and two SUMO-1 proteins, respectively. The relative amount of the sumoylated proteins increased following transfection with a UBC9 expression vector. The presence of the sumoylated form in HeLa cells solely transfected by OZF indicates the physiological activity of the endogenous SUMO-1 conjugation pathway. Using deletion mutants, we showed that two SUMO-1 modification sites are located on the sixth zinc finger. Mutation of two lysine residues greatly reduced the amount of the sumoylated form of OZF though their surrounding sequences differ from the consensus sequence reported for most proteins modified by SUMO-1 conjugation. Despite the presence of the sixth zinc finger, an OZF mutant containing zinc fingers 1–6 was not modified by SUMO-1 and failed to interact with UBC9. Addition of zinc finger 7 restored SUMO-1 modification and UBC9 interaction and provides evidence that a region downstream of the target lysines is required for interaction with UBC9, in order to achieve SUMO-1 modification. This is the first report of in vivo conjugation of a SUMO-1 protein to a Kruppel zinc finger motif. (Mol Cell Biochem 271: 215–223, 2005)     

Covalent modification of human homeodomain interacting protein kinase 2 by SUMO-1 at lysine 25 affects its stability

The HIPK2 protein is a critical regulator of apoptosis and functionally interacts with p53 to increase gene expression. Here we show that human HIPK2 is modified by sumoylation at lysine 25, as revealed by in vivo and in vitro experiments. While SUMO-1 modification of HIPK2 has no influence on its ability to phosphorylate p53 at serine 46, to induce gene expression, and to mediate apoptosis, a non-sumoylatable HIPK2 mutant displays a strongly increased protein stability. The N-terminal SUMO-1 modification site is conserved between all vertebrate HIPK2 proteins and is found in all members of the HIPK family of protein kinases. Accordingly, also human HIPK3 is modified by sumoylation.     

Novel progerin-interactive partner proteins hnRNP E1, EGF, Mel 18, and UBC9 interact with lamin A/C

The Hutchinson-Gilford progeria syndrome (HGPS or progeria) is an apparent accelerated aging disorder of childhood. Recently, HGPS has been characterized as one of a growing group of disorders known as laminopathies, which result from genetic defects of the lamin A/C (LMNA) gene. The majority of HGPS mutant alleles involve a silent mutation, c.2063C>T resulting in G608G, that generates a cryptic splicing site in exon 11 of LMNA and consequently truncates 50 amino acids near the C-terminus of pre-lamin A/C. To explore possible mechanisms underlying the development of HGPS, we began a search for proteins that would uniquely interact with progerin (the truncated lamin A in HGPS) using a yeast two-hybrid system. Four new progerin interactive partner proteins were identified that had not been previously found to interact with lamin A/C: hnRNP E1, UBC9 (ubiquitin conjugating enzyme E2I), Mel-18, and EGF1. However, using control and progeria fibroblasts, co-immunoprecipitation studies of endogenous proteins did not show differential binding affinity compared to normal lamin A/C. Thus, we did not find evidence for uniquely interacting partner proteins using this approach, but did identify four new lamin A/C interactive partners.     

The DNA topoisomerase I binding protein topors as a novel cellular target for SUMO-1 modification: characterization of domains necessary for subcellular localization and sumolation

Over the past years, modification by covalent attachment of SUMO (small ubiquitin-like modifier) has been demonstrated for of a number of cellular and viral proteins. While increasing evidence suggests a role for SUMO modification in the regulation of protein-protein interactions and/or subcellular localization, most SUMO targets are still at large. In this report we show that Topors, a Topoisomerase I and p53 interacting protein of hitherto unknown function, presents a novel cellular target for SUMO-1 modification. In a yeast two-hybrid system, Topors interacted with both SUMO-1 and the SUMO-1 conjugating enzyme UBC9. Multiple SUMO-1 modified forms of Topors could be detected after cotransfection of exogenous SUMO-1 and Topors induced the colocalization of a YFP tagged SUMO-1 protein in a speckled pattern in the nucleus. A subset of these Topors' nuclear speckles were closely associated with the PML nuclear bodies (POD, ND10). A central domain comprising Topors residues 437 to 574 was sufficient for both sumolation and localization to nuclear speckles. One SUMO-1 acceptor site at lysine residue 560 could be identified within this region. However, sumolation-deficient Topors mutants showed that sumolation obviously is not required for localization to nuclear speckles.     

Ubc9 is essential for viability of higher eukaryotic cells

Ubc9 is an enzyme involved in the conjugation of SUMO-1 (small ubiquitin related modifier 1) to target proteins. The SUMO-1 conjugation system is well conserved from yeasts to higher eukaryotes, but many SUMO-1 target proteins reported recently in higher eukaryotic cells, including IkappaBalpha, MDM2, p53, and PML, are not present in yeasts. To determine the physiological roles of SUMO-1 conjugation in higher eukaryotic cells, we constructed a conditional UBC9 mutant of chicken DT40 cells containing the UBC9 transgene under control of a tetracycline-repressible promoter and characterized their loss of function phenotypes. Ubc9 disappeared 3 days after the addition of tetracycline and the increase in viable cell number stopped 4 days after the addition of drug. In contrast to the cases of ubc9 mutants of budding and fission yeasts, which show defects in progression of G2 or early M phase and in chromosome segregation, respectively, we did not observe accumulation of cells in G2/M phase or a considerable increase in the frequency of chromosome missegregation upon depletion of Ubc9 but we did observe an increase in the number of cells containing multiple nuclei, indicating defects in cytokinesis. A considerable portion of the Ubc9-depleted cell population was committed to apoptosis without accumulating in a specific phase of the cell cycle, suggesting that chromosome damages are accumulated in Ubc9-depleted cells, and apoptosis is triggered without activating checkpoint mechanisms under conditions of SUMO-1 conjugation system impairment.     

Interaction of the developmental regulator SALL1 with UBE2I and SUMO-1

Mutations in the SALL1 gene on chromosome 16q12.1 cause Townes-Brocks syndrome (TBS). This autosomal dominantly inherited disorder is characterized by typical malformations of the thumbs, the ears, and the anus, and also commonly affects the kidneys and other organ systems. SALL1 has recently been shown to localize to chromocenters and other heterochromatin foci in murine fibroblasts and to interact with the telomere-repeat-binding factor TRF1/PIN2. Here, we show that the ubiquitin-conjugating enzyme 2I (UBE2I), the human homolog of S. cerevisiae UBC9, and the small ubiquitin-like modifier-1 (SUMO-1) interact with SALL1 in the yeast two-hybrid system. The interaction of SALL1 and UBE2I was confirmed in a glutathione S-transferase (GST) pull-down experiment. In an in vitro assay, it could be demonstrated that SALL1 is covalently modified by at least two SUMO-1 molecules in the presence of UBA2/AOS1 and UBE2I. Mutation of lysine 1086 of SALL1 to arginine abrogates SALL1 sumoylation, suggesting the presence of a polymeric SUMO-1 chain in the wild type state.