重组人Fab金属螯合层析法纯化条件的研究

在重组人Fab(rh Fab)表达载体的羧基端插入六个组氨酸, 使其对金属螯合层析介质产生特异性吸附, 可用金属螯合亲和层析法进行分离纯化. 采用自制金属(铜、锌金属离子)螯合层析介质, 以pH和咪唑两种洗脱方法,对rh Fab段的纯化效果进行了探讨. 结果显示: 铜离子螯合层析介质比锌离子螯合层析介质对rh Fab的亲和能力更强; pH洗脱方法的重复性优于咪唑法; 金属铜离子螯合层析法对rh Fab进行一步纯化可得到纯度大于95%的rh Fab产品.     

禽多杀性巴氏杆菌P1059成熟黏附蛋白的原核表达、纯化和抗原性检测

目的：建立rCPM36在大肠杆菌中的表达体系,纯化表达产物并检测其抗原性。方法：运用PCR方法从禽多杀性巴氏杆菌国际标准株P1059基因组中扩增出编码36kDa成熟黏附蛋白的cpm36基因,构建原核表达载体pQE30-cpm36,转化到大肠杆菌M15中并诱导表达目的蛋白,用镍离子螯合层析柱纯化目的蛋白及制备其抗体,Western印迹分析其抗原性。结果：SDS-PAGE结果显示目标蛋白以可溶性形式表达在大肠杆菌M15细胞质中,其相对分子质量为37kDa,Western印迹结果表明表达蛋白具有良好的抗原性。结论：成功构建出原核表达载体并实现了目的蛋白表达,用镍离子螯合层析柱纯化得到具有抗原性的蛋白,为进一步开展禽多杀性巴氏杆菌黏附因子和保护性抗原的研究奠定基础。     

琥珀酰辅酶A转移酶的表达纯化及抗体制备

目的:琥珀酰辅酶A转移酶(SCOT)是酮体代谢过程中的关键限速酶,此酶缺陷多由SCOT基因突变引起,患者多有酮症酸中毒表现。为了进一步研究SCOT的功能,采用原核表达系统表达并纯化重组SCOT,制备SCOT多克隆抗体。方法:选择蛋鸡、肉鸡模式生物为研究对象,通过生物信息学对其抗原性和属间同源性进行分析,通过RT-PCR从鸡的骨骼肌cDNA中扩增了SCOT基因N端半长片段,克隆到表达载体pET28b中,在大肠杆菌BL21(DE3)中诱导表达,并用镍离子螯合柱(Ni-NTA)纯化重组SCOT;用纯化的重组SCOT免疫小鼠后得到多克隆抗体。结果:Western印迹表明,制备的SCOT抗体具有较高的特异性,可特异性识别鸡的SCOT蛋白,同时可特异性识别小鼠和人的相应SCOT蛋白。结论:SCOT多克隆抗体的制备为后续在鸡、鼠和人中研究SCOT基因提供基础。     

致龋变异链球菌N-乙酰谷氨酸激酶的表达、纯化和生化特性研究

探讨了在大肠杆菌中实现致龋变异链球菌N-乙酰谷氨酸激酶基因(argB)的表达、蛋白纯化和生化特性研究。以变异链球菌基因组DNA为模板,设计特异引物,PCR扩增argB基因。经消化和连接构建重组载体pET28a-argB,测序确认后转化表达菌E.coli BL21(DE3)。SDS-PAGE鉴定argB基因能诱导表达,且表达物可溶。通过镍离子螯合层析和分子筛纯化成功获得N-乙酰谷氨酸激酶(NAGK)重组蛋白。NAGK酶促反应分析表明：精氨酸生物合成的乙酰化环式路径关键酶NAGK活性不受精氨酸反馈抑制,提示可能存在其他调节方式有待进一步研究。此外,分析型分子筛结果显示：具有催化活性NAGK以单体形式存在,显然不同于此前氨基酸激酶家族中的相关报道。     

抗hARD1抗血清制备及其初步肿瘤免疫组化分析

人ARD1(human arrest defective 1, hARD1)基因被确定具有N-乙酰基转移酶活性, 但其生理学功能并不清楚。为了探讨hARD1基因与肿瘤的关系, 检测hARD1蛋白在不同肿瘤中的表达, 克隆了hARD1基因并进行原核表达, 利用镍离子螯合(His-bind)柱层析纯化, 得到纯度达95%以上的hARD1蛋白。以纯化的重组蛋白免疫小鼠, 制备了抗hARD1蛋白的抗血清。利用抗hARD1多抗血清检测常见的临床肿瘤病理组织, 发现hARD1蛋白在乳腺肿瘤、前列腺癌, 以及肺癌中有较高频率的表达, 其中乳腺肿瘤中的表达频率最高, 达到70%, 远高于其他肿瘤组织。表明hARD1蛋白的高表达可能是乳腺肿瘤组织的一个标志, 为进一步揭示hARD1与乳腺肿瘤的关系奠定了基础。     

Hus1(Hus1f)基因可溶性表达、蛋白纯化及抗体制备

为进一步了解Hus1蛋白在细胞周期检查点信号传导通路中的功能 ,用RT PCR技术从NIH3T3细胞的总RNA中扩增出Hus1基因片段Hus1f(6 0 6～ 84 6bp) ,使其在大肠杆菌中呈可溶性表达 .用镍离子螯合柱 (Ni NTA)纯化Hus1f蛋白 ,获得纯度较高的蛋白 .用纯蛋白免疫小鼠制备多克隆抗体 .ELISA结果显示效价达到 1∶6 40 0 .Western印迹结果表明 ,该抗体可以与Hus1蛋白特异结合 .通过免疫共沉淀实验发现 ,Hus1蛋白与Chk1蛋白之间存在相互作用 ,为进行信号转导通路的研究奠定了基础 .     

嗜水气单胞菌十一碳焦磷酸合成酶XreF的原核表达及分离纯化

目的:构建嗜水气单胞菌十一碳焦磷酸合成酶XreF基因的原核表达载体,转入大肠杆菌诱导表达高活性的XreF蛋白。方法:从嗜水气单胞菌中提取基因组DNA,以基因组DNA为模板进行PCR扩增,构建pPROEX-HTaXreF重组载体,转入大肠杆菌BL21(DE3)表达目的蛋白;用镍离子亲和层析、离子交换层析、凝胶过滤层析分离纯化目的蛋白XreF;用分析型凝胶过滤柱检测XreF的聚集状态。结果:构建了重组载体pPROEX-HTa-XreF,测序结果与XerF基因编码序列一致;XreF蛋白在大肠杆菌中经IPTG诱导高效表达,纯化获得高浓度(17.5 mg/mL)、高纯度(96%以上)的XreF蛋白;分析型凝胶过滤结果显示,XreF在溶液中为单体,在镁离子存在的情况下为二聚体。结论:获得并纯化了在大肠杆菌中高效表达的嗜水气单胞菌XreF,镁离子可诱导XreF在溶液中由单体向二聚体转化。     

黄瓜CsERECTA基因的原核表达及其多克隆抗体制备

ERECTA基因编码一个富含亮氨酸重复序列结构的丝/苏氨酸类受体蛋白激酶,参与调控植物器官的形态建成,在株型控制及抗逆方面也有重要作用。该研究通过构建带有maltose binding protein(MBP)标签的pET21a-CsERECTA融合蛋白原核表达载体,实现了在大肠杆菌(E.coli)BL21(DE3)中的高效表达,并对诱导表达的温度、时间和IPTG浓度进行了优化。利用镍离子螯合层析纯化得到MBP-CsERECTA融合蛋白,再用rTEV蛋白酶对其进行酶切,得到CsERECTA蛋白并制备了该蛋白的多克隆抗体。结果表明,黄瓜CsERECTA蛋白以可溶和包涵体2种形式表达,低温有助于蛋白以可溶性形式大量存在。最佳诱导温度为23℃,诱导时间为6h,IPTG浓度为0.5mmol·L~(-1)。通过Western blot可检测到黄瓜内源的CsERECTA蛋白,说明制备的多可隆抗体具有较好的特异性。多克隆抗体的成功制备为进一步研究CsERECTA的功能奠定了基础。     

新型rhNDPK-A工程菌的构建及表达产物纯化研究

目的 :为简化纯化过程 ,获得有临床研究价值的rhNDPK -A蛋白 ,构建新型rhNDPK -A基因的表达质粒 ,利用 6×His标签以Ni+ -NTA亲和层析柱纯化蛋白。方法 :将抑癌基因nm2 3-H1从质粒PBVNMH1中亚克隆于带有纯化标签的表达载体pQE4 0中。IPTG诱导表达目的蛋白。通过镍离子螯合层析柱一步纯化法纯化目的蛋白。结果 :pQE - 4 0中亚克隆的nm2 3-H1序列完全正确 ;目的蛋白在大肠杆菌M15中的表达量可达 4 9.6 % ;Ni+ -NTA亲和层析柱一步纯化后蛋白纯度为 93%。结论 :构建了带有 6×His纯化标签的新型rhNDPK -A基因表达质粒pQE -nm2 3H1,所构建质粒能高效表达目的蛋白 ,利用Ni+ -NTA亲和层析柱简便高效地纯化了表达产物。     

铜绿假单胞菌LexA蛋白的纯化及免疫活性分析

目的：对铜绿假单胞菌(Pseudomonas aeruginosa,PA)的LexA蛋白进行表达、纯化,并检测其免疫活性。方法： lexA基因片段插入表达载体pET32a(+),在E.coli BL21(DE3)中表达。包涵体经洗涤并用8M尿素溶解,镍离子亲合柱层析为第一步纯化,Superdex 75凝胶过滤层析作为第二步精细纯化,HPLC测定蛋白的浓度,将纯化的LexA蛋白经注射途径免疫家兔,制备兔抗LexA血清,采用免疫双扩、ELISA及Western Blot分析LexA的免疫活性。结果：LexA以包涵体形式表达,经镍离子亲合柱层析和凝胶过滤层析二步组合纯化目的蛋白,经HPLC测定目的蛋白的最终纯度为98.97%,表达及纯化的LexA具有良好的免疫活性。     

New methods of protein purification. Affinity ultrafiltration.

This review describes a recently developed method for protein purification-affinity ultrafiltration. In affinity ultrafiltration, the protein to be purified is complexed with a macroligand composed of a soluble polymer or an insoluble microparticle with covalently bound, target protein-specific affinity ligands. The complex is trapped by an ultrafiltration membrane, whereas unwanted proteins pass through the membrane. The unwanted proteins are removed from the system by the carrier liquid. The system is then supplemented with an agent eluting the target protein by dissociating it from the microligand complex. The purified protein then passes the membrane, while the macroligand is trapped by it. The macroligand can be re-used after regeneration. Affinity ultrafiltration has a number of advantages over other protein purification techniques: 1) commercial availability of ultrafiltration systems with various high-productivity designs; 2) availability of presynthesized macroligands, which can be supplemented with additional, easily manufactured, commercial latex-based macroligands; 3) rapid separation of large solution volumes; 4) repeated use of equipment, enabling consecutive purification of different proteins; 5) simple scale-up and automation procedures.     

Hyaluronan synthase 1 (HAS1) produces a cytokine-and glucose-inducible,CD44-dependent cell surface coat

Hyaluronan is a ubiquitous glycosaminoglycan involved in embryonic development, inflammation and cancer. In mammals, three hyaluronan synthase isoenzymes (HAS1-3) inserted in the plasma membrane produce hyaluronan directly on cell surface. The mRNA level and enzymatic activity of HAS1 are lower than those of HAS2 and HAS3 in many cells, obscuring the importance of HAS1. Here we demonstrate using immunocytochemistry and transfection of fluorescently tagged HAS1 that its enzymatic activity depends on the ER–Golgi–plasma membrane traffic, like reported for HAS2 and HAS3. When cultured in 5 mM glucose, HAS1-transfected MCF-7 cells show very little cell surface hyaluronan, detected with a fluorescent hyaluronan binding probe. However, a large hyaluronan coat was seen in cells grown in 20 mM glucose and 1 mM glucosamine, or treated with IL-1β, TNF-α, or TGF-β. The coats were mostly removed by the presence of hyaluronan hexasaccharides, or Hermes1 antibody, indicating that they depended on the CD44 receptor, which is in a contrast to the coat produced by HAS3, remaining attached to HAS3 itself. The findings suggest that HAS1-dependent coat is induced by inflammatory agents and glycemic stress, mediated by altered presentation of either CD44 or hyaluronan, and can offer a rapid cellular response to injury and inflammation.     

Purification of an elastin-like fusion protein by microfiltration

This article describes a simple and potentially scalable microfiltration method for purification of recombinant proteins. This method is based on the fact that when an elastin-like polypeptide (ELP) is fused to a target protein, the inverse phase transition behavior of the ELP tag is imparted to the fusion protein. Triggering the phase transition of a solution of the ELP fusion protein by an increase in temperature, or isothermally by an increase in salt concentration, results in the formation of micron-sized aggregates of the ELP fusion protein. In this article, it is shown that these aggregates are efficiently retained by a microfiltration membrane, while contaminating E. coli proteins passed through the membrane upon washing. Upon reversing the phase transition by flow of Milli-Q water, soluble, pure, and functionally active protein is eluted from the membrane. Proof-of principle of this approach was demonstrated by purifying a fusion of thioredoxin with ELP (Trx-ELP) with greater than 95% recovery of protein and with greater than 95% purity (as estimated from SDS-PAGE gels). The simplicity of this method is demonstrated for laboratory scale purification by purifying Trx-ELP from cell lysate using a syringe and a disposable microfiltration cartridge. The potential scalability of this purification as an automated, continuous industrial-scale process is also demonstrated using a continuous stirred cell equipped with a microfiltration membrane.     

Plasma membrane residence of hyaluronan synthase is coupled to its enzymatic activity

Hyaluronan is a multifunctional glycosaminoglycan up to 10(7) Da molecular mass produced by the integral membrane glycosyltransferase, hyaluronan synthase (HAS). When expressed in keratinocytes, N-terminally tagged green fluorescent protein-HAS2 and -HAS3 isoenzymes were found to travel through endoplasmic reticulum (ER), Golgi, plasma membrane, and endocytic vesicles. A distinct enrichment of plasma membrane HAS was found in cell protrusions. The total turnover time of HAS3 was 4-5 h as judged by the green fluorescent protein signal decay and hyaluronan synthesis inhibition in cycloheximide-treated cells. The transfer from ER to Golgi took about 1 h, and the dwell time on the plasma membrane was less than 2 h in experiments with a relief and introduction, respectively, of brefeldin A. Constructs of HAS3 with 16- and 45-amino-acid C-terminal deletions mostly stayed within the ER, whereas a D216A missense mutant was localized within the Golgi complex but not the plasma membrane. Both types of mutations were almost or completely inactive, similar to the wild type enzyme that had its entry to the plasma membrane experimentally blocked by brefeldin A. Inhibition of hyaluronan synthesis by UDP-glucuronic acid starvation using 4-methyl-umbelliferone also prevented HAS access to the plasma membrane. The results demonstrate that 1) a latent pool of HAS exists within the ER-Golgi pathway; 2) this pool can be rapidly mobilized and activated by insertion into the plasma membrane; and 3) inhibition of HAS activity through mutation or substrate starvation results in exclusion of HAS from the plasma membrane.     

Determination of Biotinylated Proteins as an Index for Purification of Plasma Membrane using Surface Plasmon Resonance-based Optical Biosensor

Proteins of plasma membrane could be an index of purification of the plasma membrane of animal cells. A convenient method is proposed for determining the plasma membrane proteins by a surface plasmon resonance (SPR) biosensor. Biotinylated proteins were observed only in the peripheral areas of MOLT-4 cells which were treated by 5-[5-(N-succinimidyloxycarbonyl) pentylamido] hexyl-d-biotinamide. The proteins on HeLa cells were also biotinylated. And then the membrane samples of the HeLa cells were injected onto the avidin-immobilized SPR-surface, and components bound non-specifically on the surface were removed by a washout solution. The amount of biotinylated protein (BP) was determined directly from the absolute resonance unit (RU) after injection of the washout solution. In the method a reference surface was not needed. The amount of BP bound to the surface was gradually attenuated with the repeated injection, and a method for calibrating the RU value was introduced by considering the ratio of attenuation by every injection. The correlation between the BP titer calculated by the calibration and the theoretically-estimated one was greatly improved. Three cycles of the BP determination on a sensor surface was performed successfully. During the purification process of membrane fractions, the degree of purification as judged by the BP titer was in good agreement with the degree of increase in aminopeptidase N activity in the membrane fraction. Thus, the BP titer could be used as an index for purification of plasma membrane.     

'Piggy-back' transport of Xenopus hyaluronan synthase (XHAS1) via the secretory pathway to the plasma membrane

Hyaluronan is the sole glycosaminoglycan whose biosynthesis takes place directly at the plasma membrane. The mechanism by which hyaluronan synthase (HAS) becomes inserted there, as well as the question of how the enzyme discriminates between particular membrane species in polarized cells, are largely unknown. In vitro translation of HAS suggested that the nascent protein becomes stabilized in the presence of microsomal membranes, but would not insert spontaneously into membranes after being translated in the absence of those. We therefore monitored the membrane attachment of enzymatically active fusion proteins consisting of Xenopus HAS1 and green fluorescent protein shortly after de novo synthesis in Vero cells. Our data strongly suggest that HAS proteins are directly translated on the ER membrane without exhibiting an N-terminal signal sequence. From there the inactive protein is transferred to the plasma membrane via the secretory pathway. For unknown reasons, HAS inserted into membranes other than the plasma membrane remains inactive.     

Rab10-mediated Endocytosis of the Hyaluronan Synthase HAS3 Regulates Hyaluronan Synthesis and Cell Adhesion to Collagen

Hyaluronan synthases (HAS1–3) are unique in that they are active only when located in the plasma membrane, where they extrude the growing hyaluronan (HA) directly into cell surface and extracellular space. Therefore, traffic of HAS to/from the plasma membrane is crucial for the synthesis of HA. In this study, we have identified Rab10 GTPase as the first protein known to be involved in the control of this traffic. Rab10 colocalized with HAS3 in intracellular vesicular structures and was co-immunoprecipitated with HAS3 from isolated endosomal vesicles. Rab10 silencing increased the plasma membrane residence of HAS3, resulting in a significant increase of HA secretion and an enlarged cell surface HA coat, whereas Rab10 overexpression suppressed HA synthesis. Rab10 silencing blocked the retrograde traffic of HAS3 from the plasma membrane to early endosomes. The cell surface HA coat impaired cell adhesion to type I collagen, as indicated by recovery of adhesion following hyaluronidase treatment. The data indicate a novel function for Rab10 in reducing cell surface HAS3, suppressing HA synthesis, and facilitating cell adhesion to type I collagen. These are processes important in tissue injury, inflammation, and malignant growth.     

Effect of a cholesterol-rich lipid environment on the enzymatic activity of reconstituted hyaluronan synthase

Hyaluronan synthase (HAS) is a unique membrane-associated glycosyltransferase and its activity is lipid dependent. The dependence however is not well understood, especially in vertebrate systems. Here we investigated the functional association of hyaluronan synthesis in a cholesterol-rich membrane-environment. The culture of human dermal fibroblasts in lipoprotein-depleted medium attenuated the synthesis of hyaluronan. The sequestration of cellular cholesterol by methyl-ß-cyclodextrin also decreased the hyaluronan production of fibroblasts, as well as the HAS activity. To directly evaluate the effects of cholesterol on HAS activity, a recombinant human HAS2 protein with a histidine-tag was expressed as a membrane protein by using a baculovirus system, then successfully solubilized, and isolated by affinity chromatography. When the recombinant HAS2 proteins were reconstituted into liposomes composed of both saturated phosphatidylcholine and cholesterol, this provided a higher enzyme activity as compared with the liposomes formed by phosphatidylcholine alone. Cholesterol regulates HAS2 activity in a biphasic manner, depending on the molar ratio of phosphatidylcholine to cholesterol. Furthermore, the activation profiles of different lipid compositions were determined in the presence or absence of cholesterol. Cholesterol had the opposite effect on the HAS2 activity in liposomes composed of phosphatidylethanolamine or phosphatidylserine. Taken together, the present data suggests a clear functional association between HAS activity and cholesterol-dependent alterations in the physical and chemical properties of cell membranes.     

Integrated solid-phase synthesis and purification of PEGylated protein

We describe an integrated method for solid-phase protein PEGylation and the purification of mono-PEGylated protein thus synthesized. Lysozyme was used as model protein in this study. Methoxy-polyethyleneglycol propionaldehyde (or m-PEG propionaldehyde) was first immobilized on a stack of microporous hydrophobic interaction membranes housed in a module. The membrane-bound m-PEG propionaldehyde was then contacted with lysozyme solution, which also contained sodium cyanoborohydride as a reducing agent. The PEGylated lysozyme thus synthesized remained attached to the membrane, whereas unreacted protein could easily be removed from the module. PEGylated protein was then eluted from the membrane in a partially purified form using salt-free buffer. Two separate steps were thus integrated into a single process: protein PEGylation, followed by purification of mono-PEGylated protein. This solid-phase method is likely to be suitable for PEGylating any protein because it is based on the immobilization of the activated PEG and not the protein being PEGylated.