Expression,purification, and characterization of a novel Ca<Superscript>2+</Superscript>- and phospholipid-binding protein annexin B2

Annexin B2 (AnxB2) is a novel member of the annexin family of Ca²⁺- and phospholipid-binding proteins from Cysticercus cellulosae. To obtain highly pure AnxB2 with an easy and inexpensive purification approach, its cDNA was cloned into the prokaryotic expression vector pJLA503 and the translation initiation codon was immediately under the control of the inducible bacteriophage λ promoters P _R and P _L. After induction by shifting temperature, large amounts of non-fusion protein were produced in Escherichia coli in a soluble form. Then a novel purification method based on Ca²⁺-dependent phosphatidylserine (PS)-binding activity was established, whereby the purity of AnxB2 was increased to 98.7%. Western blot analysis showed that recombinant AnxB2 was specifically recognized by serum of pigs infected with cysticercosis. In vitro test showed that, the recombinant AnxB2 had anticoagulant activity and platelet binding activity. The expression, purification, and initial characterization of AnxB2 set an important stage for further characterization of the protein.     

人脑源性神经营养因子基因的克隆及在大肠杆菌中表达

人脑源性神经营养因子基因的克隆及在大肠杆菌中表达何晓龙,路长林,王成海（第二军医大学神经生物学教研室,上海２００４３３）关键词神经营养因子;基因克隆脑源性神经营养因子（ｂｒａｉｎ－ｄｅｒｉｖｅｄｎｅｕｒｏｔｉｃ…ｉ。血。ｔｏｒ,ＢＤ贾助是Ｂａｄｅ等人...     

Construction and Co-expression of Grass Carp Reovirus VP6 Protein and Enhanced Green Fluorescence Protein in the Insect Cells

Grass carp reovirus(GCRV),a disaster agent to aquatic animals,belongs to Genus Aquareovirus of family Reoviridea.Sequence analysis revealed GCRV genome segment 8(s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa.To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter,the recombinant baculovirus,which contained the GCRVs8 and eGFP(enhanced green fluorescence protein) genes,was constructed by using the Bac-to-Bac insect expression system.In this study,the whole GCRVs8 and eGFP genes,amplified by PCR,were constructed into a pFastBacDual vector under polyhedron(PH) and p10 promoters,respectively.The constructed dual recombinant plasmid(pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid(AcGCRVs8/eGFP) by transposition.Finally,the recombinant bacluovirus(vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells.The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection,and gradually enhanced and extended around 5 days culture in P1(Passage1) stock.The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus(BV) stock.Additionally,PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus.Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro.     

Construction and co-expression of grass carp reovirus VP6 protein and enhanced green fluorescence protein in the insect cells

Grass carp reovirus (GCRV), a disaster agent to aquatic animals, belongs to Genus Aquareovirus of family Reoviridea. Sequence analysis revealed GCRV genome segment 8 (s8) was 1 296 bp nucleotides in length encoding an inner capsid protein VP6 of about 43kDa. To obtain in vitro non-fusion expression of a GCRV VP6 protein containing a molecular of fluorescence reporter, the recombinant baculovirus, which contained the GCRVs8 and eGFP (enhanced green fluorescence protein) genes, was constructed by using the Bac-to-Bac insect expression system. In this study, the whole GCRVs8 and eGFP genes, amplified by PCR, were constructed into a pFastBacDual vector under polyhedron (PH) and p10 promoters, respectively. The constructed dual recombinant plasmid (pFbDGCRVs8/eGFP) was transformed into DH10Bac cells to obtain recombinant Bacmid (AcGCRVs8/eGFP) by transposition. Finally, the recombinant bacluovirus (vAcGCRVs8/eGFP) was obtained from transfected Sf9 insect cells. The green fluorescence that was expressed by transfected Sf9 cells was initially observed 3 days post transfection, and gradually enhanced and extended around 5 days culture in P1(Passage1) stock. The stable high level expression of recombinant protein was observed in P2 and subsequent passage budding virus (BV) stock. Additionally, PCR amplification from P1 and amplified P2 BV stock further confirmed the validity of the dual-recombinant baculovirus. Our results provide a foundation for expression and assembly of the GCRV structural protein in vitro. Undergraduate training student from College of Life Sciences, Wuhan University.     

油菜AP2/ERF-B4类转录因子克隆及表达载体的构建

利用油菜UniGene数据库,以拟南芥转录因子保守序列为探针,通过电子克隆方法分离得到一个UniGene库Bna.17538,进一步序列拼接得到一个油菜AP2/ERF-B4亚族的转录因子BnaERFB4-1,长度为672 bp,并进行了相关的生物信息学分析.结果显示BnaERFB4-1是亲水性蛋白,蛋白质三级结构与拟南芥RAP2.6L非常相似,蛋白质无序化程度大于拟南芥RAP2.6L.设计引物通过PCR和RT-PCR方法分别从甘蓝型油菜沪油15幼苗的DNA和cDNA中分离了BnaERFB4-1基因,命名为BnaERFB4-1-Hy15.序列测定和分析显示,来源于沪油15的BnaERFB4-1-Hy15基因与电子克隆的基因序列差异很小,有3个氨基酸位点不同,存在一个内含子.将BnaERFB4-1-Hy15基因通过BamHⅠ和SacⅠ酶切后分别插入酵母表达载体YK3302和植物双元表达载体pYF1404的相应位置,构建了BnaERFB4-1-Hy15基因的酵母体内结合和植物转化载体,为深入研究该基因在油菜抗逆调控中的作用奠定了基础.     

核酸酶P1的原核表达、纯化及酶学特性分析

为了建立一种核酸酶P1(Nuclease P1,NP1)的原核表达纯化系统,首先采用重叠延伸PCR将22段寡核苷酸拼接,获得人工合成的NP1基因。将其克隆至分泌型表达载体pMAL-p4X获得重组质粒pMAL-p4X-NP1,然后将重组载体转化T7 Express和Origami B(DE3)菌株诱导表达,利用Amylose亲和层析柱纯化获得重组蛋白,并对其活性、热稳定性和金属离子依赖性进行系统分析。SDS-PAGE结果显示,重组蛋白MBP-NP1(Maltose binding protein-NP1)在T7 Express和Origami B(DE3)菌株中均可表达,且以可溶性形式存在。活性检测表明Origami B(DE3)菌株中获得的重组蛋白活性高于T7 Express菌株(75.48 U/mg:51.50 U/mg);利用蛋白酶Factor Xa切除MBP标签后,两种重组蛋白的比活力均有提高,分别为258.13 U/mg和139.20 U/mg。重组NP1表现出良好的热稳定性,80℃温浴30 min后重组酶仍具有90%以上的活力。2.0 mmol/L Zn2+对NP1有比较明显的激活作用,相同浓度的Cu2+则对该酶有强烈的抑制作用。该研究实现了NP1在大肠杆菌系统中的功能性表达,为NP1纯酶的制备提供一个替代途径。     

HBcAg基因在甲醇型酵母中的表达、产物纯化及其性质的初步研究

为研究乙肝核心抗原蛋白(HBcAg)在甲醇型酵母(PichiaPastoris)中的表达和性质,用PCR方法将HBcAg基因(L)克隆到P.Pastoris胞内表达载体pPIC3k中,并利用电击和同源重组法,将重组质粒pPIC3kL转化感受态的甲醇型GS115酵母菌株。经过筛选得到阳性P.Pastoris重组子。重组菌株经甲醇诱导培养,表达产物的Western印迹结果表明,HBcAg蛋白能在甲醇型酵母(PichiaPastoris)中诱导表达,产物为一215kDa的蛋白。经蔗糖密度梯度超离心和CsCl密度梯度超离心纯化后,ELISA和密度测定结果表明重组HBcAg蛋白主要分布在密度为12576gml和13013gml的2个峰值处。电镜观察表明,该重组HBcAg蛋白能自主装配成大小不同的2种颗粒(即核心颗粒),大颗粒直径约34nm左右,小颗粒直径约30nm左右。同时,我们还观察到,该核心蛋白颗粒在体外可发生集聚现象。     

Expression, purification, and characterization of recombinant human soluble BAFF secreted from the yeast Pichia pastoris

The B lymphocyte stimulator (BAFF) is a novel member of the tumor necrosis factor (TNF) ligand family which is important in B lymphocyte maturation and survival. Herein, the cDNA coding for the extracellular domain of the BAFF (hsBAFF) has been cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and Western blotting assays of culture broth from a methanol-induced expression strain demonstrated that recombinant hsBAFF, a 20.2 kDa glycosylated protein, was secreted into the culture medium. The recombinant protein was purified to greater than 95% using DEAE-Sepharose ion exchange and Superdex 75 size-exclusion chromatography steps. Finally, 102 mg of the protein was obtained in high purity from 1 L of the supernatant and its identity to hsBAFF was confirmed by NH(2)-terminal amino acid sequence analysis Bioactivity of the recombinant hsBAFF was confirmed by the ability of the protein to stimulate human B lymphocyte proliferation in vitro. Our results suggest that the P. pastoris expression system can be used to produce large quantities of fully functional hsBAFF for both research and industrial purpose.     

Expression, purification, and characterization of functional recombinant human daintain/AIF-1 in Escherichia coli

Daintain/AIF-1 was identified from injured rat carotid arteries and porcine intestine in the mid 1990s. It is involved in autoimmune disorders, chronic rejection of allografts, gliomas, and breast cancer. Since it is convenient and economical to obtain such a peptide biologically, in this study, we describe the expression, purification, and characterization of recombinant human daintain/AIF-1 (rhdaintain/AIF-1). The backbone of vector pET32a, a high-level expression plasmid, was used to construct the pET32a-daintain/AIF-1 plasmid for daintain/AIF-1 expression in Escherichia coli. The recombinant daintain/AIF-1 protein was solubly expressed in the BL21 (DE3) strain and was purified by Ni2+ affinity chromatography. After purification, the recombinant protein showed the expected size of 18 kDa on Tricine-SDS-PAGE gels which was further confirmed by Western blotting. A total of 34.0 mg of high purity (over 98%) rhdaintain/AIF-1 was obtained from 1 L culture. The recombinant peptide was able to increase blood glucose elimination rates and enhance the proliferation of human MCF-7 cells. These results suggest that biological activity of the recombinant peptide was preserved after purification.     

High-level expression, purification, and characterization of recombinant type A botulinum neurotoxin light chain

Botulinum neurotoxin light chain (BoNT LC, 50 kDa) is responsible for the zinc endopeptidase activity specific for proteins of neuroexocytosis apparatus. We describe the expression of recombinant type A BoNT LC in Escherichia coli as well as the purification and characterization of the recombinant protein. A high level of expression of BoNT/A LC was obtained by an extended postinduction time of 15 h at 30 degrees C. Recombinant BoNT/A LC was isolated from an Ni(2+) column. Due to its high pI ( approximately 8.7), purification was achieved by a single step of passing the protein through anion-exchange chromatography at pH 8.0 without the need of elution. The purified recombinant BoNT/A LC retained proteolytic activity and had a secondary structure similar to that of native LC determined by CD measurement.     

Identification,localization, and functional implications of an abundant nematode annexin

Cultures of the nematode C. elegans were examined for the presence of calcium-dependent, phospholipid-binding proteins of the annexin class. A single protein of apparent mass on SDS-polyacrylamide gels of 32 kD was isolated from soluble extracts of nematode cultures on the basis of its ability to bind to phospholipids in a calcium-dependent manner. After verification of the protein as an annexin by peptide sequencing, an antiserum to the protein was prepared and used to isolate a corresponding cDNA from an expression library in phage lambda gt11. The encoded protein, herein referred to as the nex-1 annexin, has a mass of 35 kD and is 36-42% identical in sequence to 10 known mammalian annexins. Several unique modifications were found in the portions of the sequence corresponding to calcium-binding sites. Possible phosphorylation sites in the NH2-terminal domain of the nematode annexin correspond to those of mammalian annexins. The gene for this annexin (nex-1) was physically mapped to chromosome III in the vicinity of the dpy-17 genetic marker. Two other annexin genes (nex-2 and nex-3) were also identified in chromosome III sequences reported by the nematode genomic sequencing project (Sulston, J., Z. Du, K. Thomas, R. Wilson, L. Hillier, R. Staden, N. Halloran, P. Green, J. Thierry-Mieg, L. Qiu, et al. 1992. Nature (Lond.). 356:37-41). The nex-1 annexin was localized in the nematode by immunofluorescence and by electron microscopy using immunogold labeling. The protein is associated with membrane systems of the secretory gland cells of the pharynx, with sites of cuticle formation in the grinder in the pharynx, with yolk granules in oocytes, with the uterine wall and vulva, and with membrane systems in the spermathecal valve. The presence of the annexin in association with the membranes of the spermathecal valve suggests a novel function of the protein in the folding and unfolding of these membranes as eggs pass through the valve. The localizations also indicate roles for the annexin corresponding to those proposed in mammalian systems in membrane trafficking, collagen deposition, and extracellular matrix formation.     

分泌型乙型肝炎病毒包膜M蛋白在毕赤酵母中的表达

将乙肝病毒包膜中蛋白M基因插入酵母整合型表达质粒pAO818的醇氧化酶 (AOX1)启动子下游 ,构建携带 8拷贝M表达盒的重组载体 ,经电穿孔转化SMD116 8菌株和G4 18筛选 ,得到了高效分泌表达M蛋白的毕赤酵母菌株 ,表达量超过 5 0mg L .经初步纯化 ,对表达产物的性质鉴定表明 ,重组蛋白具有preS2和S抗原性 ,可以形成颗粒 ,并具有一定程度的糖基化 .所构建的稳定重组菌株和所得到的重组蛋白颗粒 ,为进一步研究新一代的疫苗提供了必要的材料 .     

Production of biologically active recombinant annexin B1 with enhanced stability via a tagging system

Annexin B1 is a novel Ca²⁺-dependent phospholipid-binding protein from metacestodes of Taenia solium and has been shown to have many potential biomedical applications. Although annexin B1 has been produced successfully in Escherichia coli, the purified protein has poor stability at room temperature, which has hindered our attempts to further study its structure–function relationship. To increase the stability of the protein, the construction and purification procedures were examined and changed to hopefully increase its effectiveness. In this study, we describe a new recombinant annexin B1 expressed with a hexahistidine tag fused to its N-terminal end, which was purified to homogeneity in two steps using immobilized metal affinity followed by size exclusion chromatography. The final yield was approximately 23 mg/L of bacterial culture. Isoelectric focusing and mass spectrometry analysis showed that the protein purified by this method was quite stable at room temperature, even greater than 3 days later. A series of functional tests indicated that the recombinant protein had high anticoagulant activity, and fluorescence-labeled annexin B1 could bind to the outer membranes of apoptotic mammalian cells and efficiently detect them in the early stages of apoptosis.     

A novel,cheap and effective fusion expression system for the production of recombinant proteins

To develop faster, less expensive methods for expression and purification of proteins, the annexin B1-intein fusion expression system was constructed. The interest proteins fused to the annexin B1-intein tag were purified in a single-step method based on the Ca²⁺-binding activity of annexin B1, and the annexin B1-intein fusion tag was removed based on the self-cleaving activity of the intein. Moreover, we found that in some cases, fusion to annexin B1 can promote the solubility of heterologous proteins. The production of soluble and highly active of interleukin-2 and low-molecular single-chain urokinase in our results proved that the system was a novel, cheap and effective fusion expression system for the production of valuable soluble recombinant proteins in Escherichia coli.     

Identification and characterization of a novel Ribose 5-phosphate isomerase B from Leishmania donovani

Leishmaniasis is a group of tropical diseases caused by protozoan parasites of the genus Leishmania. Due to the emergence of resistance to the available antileishmanial drugs there is an immediate need to identify molecular targets on which to base future treatment strategies. Ribose 5-phosphate isomerase (Rpi; EC 5.3.1.6) is a key enzyme of the pentose phosphate pathway (PPP) which catalyses the reversible aldose-ketose isomerization between Ribose 5-phosphate (R5P) and Ribulose 5-phosphate (Ru5P). It exists in two isoforms A and B. These two are completely unrelated enzymes catalyzing the same reaction. Analysis of the Leishmania infantum genome revealed that though the RpiB gene is present, RpiA homologs are completely absent. An absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for the chemotherapy of Leishmaniasis. In this paper, we report for the first time the presence of B isoform of the Rpi enzyme in Leishmania donovani (LdRpiB) by cloning and molecular characterization of the enzyme. An amplified L. donovani RpiB gene is 519 bp and encodes for a putative 172 amino acid protein with a molecular mass of ～19 kDa. An ～19 kDa protein with poly-His tag at the C-terminal end was obtained by heterologous expression of LdRpiB in Escherichia coli. The recombinant form of RpiB was obtained in soluble and active form. The LdRpiB exists as a dimer of dimers i.e. the tetramer form. The polyclonal antibody against Trypanosoma cruzi RpiB could detect a band of ～19 kDa with the purified recombinant RpiB as well as native RpiB from the L. donovani promastigotes. Recombinant RpiB obeys the classical Michaelis-Menten kinetics utilizing R5P as the substrate with a K(m) value of 2.4±0.6 mM and K(cat) value of 30±5.2 s(-1). Our study confirms the presence of Ribose 5-phosphate isomerase B in L. donovani and provides functional characterization of RpiB for further validating it as a potential drug target.     

Production of human papillomavirus type 33 L1 major capsid protein and virus-like particles from Bacillus subtilis to develop a prophylactic vaccine against cervical cancer

We developed a bacterial expression system to produce human papillomavirus (HPV) type 33 L1 major capsid protein and virus-like particles from a recombinant Bacillus subtilis strain. For the first time, we have isolated self-assembled virus-like particles (VLPs) of HPV type 33 from B. subtilis, a strain generally recognized as safe (GRAS). The gene encoding the major capsid protein L1 of HPV type 33 was amplified from viral DNA isolated from a Korean patient and expressed in B. subtilis; a xylose-induction system was used to control gene activity. HPV33 L1 protein was partially purified by 40% (w/v) sucrose cushion centrifugation and strong cation exchange column chromatography. Eluted samples exhibited immunosignaling in fractions of 0.5-1.0 M NaCl. The HPV33 L1 protein was shown to be approximately 56 kDa in size by SDS-PAGE and Western blotting; recovery and purity were quantified by indirect immuno-ELISA assay. The final yield and purity were approximately 20.4% and 10.3%, respectively. Transmission electron microscopic analysis of fractions immunoactive by ELISA revealed that the L1 protein formed self-assembled VLPs with a diameter of approximately 20-40 nm. Humoral and cellular immune responses provoked by the B. subtilis/HPV33 L1 strain were approximately 100- and 3-fold higher than those of the empty B. subtilis strain as a negative control, respectively. Development of a VLP production and delivery system using B. subtilis will be helpful, in that the vaccine may be convenient production as an antigen delivery system. VLPs thus produced will be safer for human use than those purified from Gram-negative strains such as Escherichia coli. Also, use of B. subtilis as a host may aid in the development of either live or whole cell vaccines administered by antigen delivery system.     

HSV-IgM人-鼠嵌合抗体制备及稳定细胞株构建工艺开发

为实现体外大规模制备单纯疱疹病毒HSV-IgM(HSV1,HSV2)人鼠嵌合抗体,本研究通过RNA连接酶介导的cDNA末端快速扩增(RNA ligase-mediated rapid amplification of cDNA ends,RLM-RACE)技术获取其对应杂交瘤细胞基因序列,构建嵌合抗体至真核表达载体,在CHO-S细胞中稳定表达所需目的蛋白。同时优化稳定细胞株筛选工艺,对细胞池构建阶段和单克隆筛选阶段的加压条件进行摸索与探究,最后目的抗体采用蛋白L亲和纯化法进行纯化并进行生物活性检测;最终成功制备899 kDa和909 kDa的稳定高表达重组IgM抗体(HSV1,HSV2)细胞株。结果表明,最适筛选压力为20P200M(一轮加压)和50P1000M(二轮加压);使用加压培养基进行单克隆筛选抗体表达量较高,HSV1-IgM和HSV2-IgM单克隆最终表达量分别为1620 mg/L和623 mg/L。本研究为HSV1和HSV2的IgM系列重组抗体质控品开发以及体外高表达分泌IgM亚型抗体提供理论与实践基础。     

Annexin B1 from Taenia solium metacestodes is a newly characterized member of the annexin family

We previously reported cloning of the Taenia solium annexin B1 gene from a metacestode cDNA expression library and demonstrated that it acts as a protective antigen for effective vaccine development against cysticercosis. In the present study we produced recombinant annexin B1 and antiserum against the protein to investigate its structural and functional properties. Western blotting of metacestode fractions indicated that T. solium annexin B1, similar to vertebrate annexins, associates with acid phospholipids in the presence of Ca(2+). This property was confirmed by the recognition of apoptotic cells by labeled annexin B1. CD spectroscopy results demonstrated that alpha-helices are the main secondary structures of the protein. Ca(2+) binding increases the alpha-helix content and causes significant thermal stabilization with a melting temperature increase of approximately 10 degrees C. Functional Ca(2+)-dependent phospholipid binding sites of annexin B1 were investigated using mutant proteins. By changing a conserved acidic amino acid residue that putatively combines Ca(2+) in each domain of annexin B1 singly or in combination, we found that Ca(2+) binding in the first domain is more important than that at the other Ca(2+) binding sites. Annexin B1 is a metacestode stage-specific antigen, with the protein being mainly localized in the teguments and surrounding cyst wall of T. solium metacestodes, suggesting a role in the parasite-host interaction.     

Expression, purification, and inhibitory activities of mouse cytotoxic T-lymphocyte antigen-2alpha

Cytotoxic T-lymphocyte antigen-2 (CTLA-2) is a novel cysteine proteinase inhibitor. The protein sequence is homologous to the proregion of mouse cathepsin L. Here, we report the expression, purification, and characterization of recombinant CTLA-2 (CTLA-2alpha). CTLA-2alpha was cloned into the pET16b vector and the plasmid was transformed into Escherichia coli strain BL21 (DE3) pLysS. The recombinant CTLA-2alpha was highly expressed and purified by His-Bind affinity chromatography, Factor Xa digestion, and hydrophobic chromatography. Throughout these procedures, 3mg recombinant CTLA-2alpha was obtained from 450 ml of bacterial culture medium. The purified protein exhibited inhibitory activities towards certain cysteine proteinases and was properly refolded, as indicated by circular dichroism spectroscopy. Recombinant CTLA-2alpha fully inhibited Bombyx cysteine proteinase (BCP) (overall Kd (Ki*) = 0.23 nM) and and cathepsin L (overall Kd (Ki*) = 0.38 nM). Inhibition of cathepsin H ( Ki = 86 nM) and papain ( Ki = 560 nM) was much weaker, while inhibition of cathepsin B was negligible ( Ki > 1 microM). Our results indicate that mouse CTLA-2alpha is a selective inhibitor of the cathepsin L-like cysteine proteinases.     

Haemonchus contortus: prokaryotic expression and enzyme activity of recombinant HcSTK, a serine/threonine protein kinase

Members of the PAR-1/MARK serine/threonine protein kinase (STK) subfamily are important regulators of the cytoskeleton, and their characterization can provide insights into a number of critical processes relating to the development and survival of an organism. We previously investigated the mRNA expression for and organization of a gene (hcstk) representing HcSTK, an STK from the parasitic nematode Haemonchus contortus. In the present study, a recombinant form of HcSTK was expressed and characterized. Affinity-purified anti-HcSTK antibodies reacted with native HcSTK in protein homogenates extracted from third-stage larvae (L3) of H. contortus and were also used to immunolocalize the protein around the nuclei of ovarian and intestinal tissues of adult H. contortus. The enzyme activity of the recombinant HcSTK protein was also demonstrated. The findings show that recombinant HcSTK is a functional protein kinase, with activity directed to KXGS motifs, consistent with other members of the PAR-1/MARK STK subfamily.