一种抗菌肽和aFGF融合蛋白的构建和表达

利用PCR技术扩增出带有凝血酶Xa因子切割位点的天蚕素蜂毒素杂合肽和aFGF的融合基因,插入大肠杆菌表达载体pET-3c中,构建出表达质粒pET-aF-CM,并转化至大肠杆菌BL21（DE3）中,氨苄青霉素抗性筛选重组转化子。IPTG诱导4h后,以包涵体形式表达的融合蛋白约占菌体总蛋白的17%。将包涵体溶解后透析复性,并利用肝素亲和层析纯化,得到电泳纯的融合蛋白。Western blot分析表明,该蛋白能与aFGF抗体产生免疫反应。MTT法检测显示,融合蛋白具有促3T3Bal/b细胞分裂活性,其比活为1.471×106IU/mg。利用凝血酶Xa因子裂解融合蛋白,可以获得抗菌肽和含凝血酶Xa因子裂解序列的aFGF蛋白。分子筛回收含杂合抗菌肽,抑菌活性检测表明其对大肠杆菌K12D31具有明显抑菌活性。微量稀释法检测结果表明,回收的抗菌肽对大肠杆菌DH5α、大肠杆菌K12D31、沙门氏菌、金黄色葡萄球菌、枯草芽孢杆菌和绿脓杆菌的MIC分别达6.25μg/ml、10μg/ml、2.5μg/ml、1.25μg/ml、0.625μg/ml和5μg/ml。     

Construction and characterization of a bi-functional EGFP/sBAFF fusion protein

A fusion between gene encoding fluoresce-enhanced green fluorescent protein variant (EGFP) and soluble domain of human B-cell-activating factor of the TNF family (sBAFF) was constructed and expressed in Escherichia coli. The EGFP/sBAFF had an apparent molecular weight of 45 kDa and was detected with anti-hsBAFF and anti-His(6) monoclonal antibodies. After being purified by immobilized metal affinity chromatography (IMAC), the fusion protein retained similar fluorescence spectra to those of EGFP. Biological activity assays showed the EGFP/sBAFF as well as sBAFF could co-stimulated human B lymphocyte proliferation in vitro. In addition, EGFP/sBAFF has shown specific binding to BAFF receptors positive-cells and the stained cells could be analyzed with flow cytometry. Thus, the fusion protein represents a readily obtainable source of biologically active sBAFF that may prove useful in further studies on BAFF and its receptors.     

Bacterial protein toxins targeting rho GTPases

Several bacterial protein toxins target eukaryotic cells by modulating the functions of Rho GTPases that are involved in various signal processes and in the regulation of the actin cytoskeleton. The toxins inhibit Rho functions by ADP-ribosylation or glucosylation and activate them by deamidation and transglutamination. New findings indicate that the GTPases are also targeted by various 'injected' toxins which are introduced into the eukaryotic cells by the type-III secretion system. The injected toxins do not covalently modify Rho GTPases, but manipulate their regulatory GTPase cycle by acting as GTPase-activating proteins or guanine nucleotide exchange factors.     

Structure and function of a paramyxovirus fusion protein

Paramyxoviruses initiate infection by attaching to cell surface receptors and fusing viral and cell membranes. Viral attachment proteins, hemagglutinin-neuraminidase (HN), hemagglutinin (HA), or glycoprotein (G), bind receptors while fusion (F) proteins direct membrane fusion. Because paramyxovirus fusion is pH independent, virus entry occurs at host cell plasma membranes. Paramyxovirus fusion also usually requires co-expression of both the attachment protein and the fusion (F) protein. Newcastle disease virus (NDV) has assumed increased importance as a prototype paramyxovirus because crystal structures of both the NDV F protein and the attachment protein (HN) have been determined. Furthermore, analysis of structure and function of both viral glycoproteins by mutation, reactivity of antibody, and peptides have defined domains of the NDV F protein important for virus fusion. These domains include the fusion peptide, the cytoplasmic domain, as well as heptad repeat (HR) domains. Peptides with sequences from HR domains inhibit fusion, and characterization of the mechanism of this inhibition provides evidence for conformational changes in the F protein upon activation of fusion. Both proteolytic cleavage of the F protein and interactions with the attachment protein are required for fusion activation in most systems. Subsequent steps in membrane merger directed by F protein are poorly understood.     

Development of in vitro assays for the detection of botulinum toxins in foods

Currently the only accepted method for the detection of botulinum neurotoxin in contaminated samples is the mouse bioassay. Although highly sensitive this test has a number of drawbacks: it is expensive to perform, lacks specificity and involves the use of animals. With increasing resistance to such animal tests there is a need to replace the bioassay with a reliable in vitro test. Over the past six years it has been demonstrated that all the botulinum neurotoxins act intracellularly as highly specific zinc endoproteases, cleaving proteins involved in the control of secretion of neurotransmitters. In the work described, this enzymatic activity has been utilised in assay formats for the detection in foods of neurotoxin of the serotypes involved in food-borne outbreaks in man. These assays have been shown to have a greater sensitivity, speed and specificity than the mouse bioassay. It is envisaged that such assays will prove realistic alternatives to animal-based tests.     

His-FemA融合蛋白表达载体的构建及其在原核细胞中的表达

目的 构建His标签的金黄色葡萄球菌甲氧西林耐药相关蛋白FemA的融合蛋白表达载体,并在大肠埃希菌中表达,为进一步研究femA基因的生物学功能和临床应用奠定基础.方法 根据GenBank中金黄色葡萄球菌femA基因序列,利用Primer Premier 5.0设计PCR引物,并在引物的5'加入BamHI及SalI酶切位点;以金黄色葡萄球菌基因组DNA为模版,PCR扩增出femA基因片段.将目的DNA片段及质粒pQE30分别进行双酶切、连接并转化大肠埃希菌DH5α;阳性克隆以PCR、双酶切及测序进行鉴定.将鉴定正确的pQE30-femA重组质粒转化大肠埃希菌JM109,异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达His-femA融合蛋白;采用SDS-PAGE及Western blot分析对表达蛋白进行验证.结果 经PCR、双酶切签定及序列测定,证实重组质粒pQE30-femA构建成功;重组质粒pQE30-femA转化大肠埃希菌JM109经IPTG诱导后,SDS-PAGE和Western blot分析显示表达出53 kD目的蛋白;经Bandscan软件分析,目的蛋白质在4h的表达量占细胞总蛋白的27.5％.结论 成功构建了His-FemA原核表达载体(pQE30-femA),并在大肠埃希菌中高效表达.     

Immuno-rolling circle amplification using a multibinding fusion protein

Ultrasensitive detection of specific, low level proteins in body fluids is particularly challenging. Owing to the extreme sensitivity of the polymerase chain reaction step, the requirements for immuno-rolling circle amplification (immuno-RCA) are much more stringent than for conventional ELISA. Here, we report the development of a rolling circle amplification procedure using multibinding fusion protein to enhance signals of immuno-RCA to detect a cancer biomarker, α-fetoprotein (AFP). We successfully avoid the covalent linkage between antibody and DNA or antibody and biotin/streptavidin by introducing a new genetically engineered fusion protein which contains the C2 domain of protein G and biotin acceptor peptide (BAP) which is intended to maintain the biological activity of the antibody. The purified fusion protein retained its binding affinity with IgG and streptavidin after efficient expression in Escherichia coli. Immuno-RCA in combination with BAP-C2 specifically and sensitively detected AFP in a microplate format. Therefore, the sensitivity and convenient nature of this method should contribute to effective signal enhancement in immunoassays for cancer biomarker detection.     

Quantification of small GTPase glucosylation by clostridial glucosylating toxins using multiplexed MRM analysis

Large clostridial toxins mono‐O‐glucosylate small GTPases of the Rho and Ras subfamily. As a result of glucosylation, the GTPases are inhibited and thereby corresponding downstream signaling pathways are disturbed. Current methods for quantifying the extent of glucosylation include sequential [¹⁴C]glucosylation, sequential [³²P]ADP‐ribosylation, and Western Blot detection of nonglucosylated GTPases, with neither method allowing the quantification of the extent of glucosylation of an individual GTPase. Here, we describe a novel MS‐based multiplexed MRM assay to specifically quantify the glucosylation degree of small GTPases. This targeted proteomics approach achieves a high selectivity and reproducibility, which allows determination of the in vivo substrate pattern of glucosylating toxins. As proof of principle, GTPase glucosylation was analyzed in CaCo‐2 cells treated with TcdA, and glucosylation kinetics were determined for RhoA/B, RhoC, RhoG, Ral, Rap1, Rap2, (H/K/N)Ras, and R‐Ras2.     

Inhibition of the glucosyltransferase activity of clostridial Rho/Ras-glucosylating toxins by castanospermine

Castanospermine was identified as an inhibitor of the Rho/Ras-glucosylating Clostridium sordellii lethal toxin and Clostridium difficile toxin B. Microinjection of castanospermine into embryonic bovine lung cells prevented the cytotoxic effects of toxins. The crystal structure of the glucosyltransferase domain of C. sordellii lethal toxin in complex with castanospermine, UDP and a calcium ion was solved at a resolution of 2.3A. The inhibitor binds in a conformation that brings its four hydroxyl groups and its N-atom almost exactly in the positions of the four hydroxyls and of the ring oxygen of the glucosyl moiety of UDP-glucose, respectively.     

Conformational changes and reaction of clostridial glycosylating toxins

The crystal structures of the catalytic fragments of ‘lethal toxin’ from Clostridium sordellii and of ‘α-toxin’ from Clostridium novyi have been established. Almost half of the residues follow the chain fold of the glycosyl-transferase type A family of enzymes; the other half forms large α-helical protrusions that are likely to confer specificity for the respective targeted subgroup of Rho proteins in the cell. In the crystal, the active center of α-toxin contained no substrates and was disassembled, whereas that of lethal toxin, which was ligated with the donor substrate UDP-glucose and cofactor Mn^2 +, was catalytically competent. Surprisingly, the structure of lethal toxin with Ca^2 + (instead of Mn^2 +) at the cofactor position showed a bound donor substrate with a disassembled active center, indicating that the strictly octahedral coordination sphere of Mn^2 + is indispensable to the integrity of the enzyme. The homologous structures of α-toxin without substrate, distorted lethal toxin with Ca^2 + plus donor, active lethal toxin with Mn^2 + plus donor and the homologous Clostridium difficile toxin B with a hydrolyzed donor have been lined up to show the geometry of several reaction steps. Interestingly, the structural refinement of one of the three crystallographically independent molecules of Ca^2 +-ligated lethal toxin resulted in the glucosyl half-chair conformation expected for glycosyl-transferases that retain the anomeric configuration at the C1″ atom. A superposition of six acceptor substrates bound to homologous enzymes yielded the position of the nucleophilic acceptor atom with a deviation of < 1 Å. The resulting donor-acceptor geometry suggests that the reaction runs as a circular electron transfer in a six-membered ring, which involves the deprotonation of the nucleophile by the β-phosphoryl group of the donor substrate UDP-glucose.     

Structural characterization of mumps virus fusion protein core

The fusion proteins of enveloped viruses mediating the fusion between the viral and cellular membranes comprise two discontinuous heptad repeat (HR) domains located at the ectodomain of the enveloped glycoproteins. The crystal structure of the fusion protein core of Mumps virus (MuV) was determined at 2.2 A resolution. The complex is a six-helix bundle in which three HR1 peptides form a central highly hydrophobic coiled-coil and three HR2 peptides pack against the hydrophobic grooves on the surface of central coiled-coil in an oblique antiparallel manner. Fusion core of MuV, like those of simian virus 5 and human respiratory syncytium virus, forms typical 3-4-4-4-3 spacing. The similar characterization in HR1 regions, as well as the existence of O-X-O motif in extended regions of HR2 helix, suggests a basic rule for the formation of the fusion core of viral fusion proteins.     

Heterogeneity of large clostridial toxins: importance of Clostridium difficile toxinotypes

Clostridium difficile toxinotypes are groups of strains defined by changes in the PaLoc region encoding two main virulence factors: toxins TcdA and TcdB. Currently, 24 variant toxinotypes (I-XXIV) are known, in addition to toxinotype 0 strains, which contain a PaLoc identical to the reference strain VPI 10463. Variant toxinotypes can also differ from toxinotype 0 strains in their toxin production pattern. The most-studied variant strains are TcdA-, TcdB+ (A-B+) strains and binary toxin CDT-producing strains. Variations in toxin genes are also conserved on the protein level and variant toxins can differ in size, antibody reactivity, pattern of intracellular targets (small GTPases) and consequently in their effects on the cell. Toxinotypes do not correlate with particular forms of disease or patient populations, but some toxinotypes (IIIb and VIII) are currently associated with disease of increased severity and outbreaks worldwide. Variant toxinotypes are very common in animal hosts and can represent from 40% to 100% of all isolates. Among human isolates, variant toxinotypes usually represent up to 10% of strains but their prevalence is increasing.     

融合表达氨基转移酶DsaD和异构酶DsaE合成L-别异亮氨酸

【背景】L-异亮氨酸(L-isoleucine,L-Ile)和L-别异亮氨酸(L-allo-isoleucine,L-allo-Ile)是自然界中广泛存在的一对同分异构体。抗感染抗生素Desotamides结构中含L-别异亮氨酸结构单元,其生物合成途径中的氨基转移酶DsaD和异构酶DsaE可以协作催化L-异亮氨酸和L-别异亮氨酸相互转化。【目的】通过理性设计,使氨基转移酶DsaD和异构酶DsaE融合表达,研究融合蛋白DsaDE催化异亮氨酸和别异亮氨酸相互转化的功能。【方法】利用PCR分别扩增dsaE基因编码区DNA片段、以及含dsaD基因编码区和114个碱基接头序列的DNA片段dsaD-linker,利用酶切位点KpnI将dsaE和dsaD-linker相连,形成das DE重组序列,并克隆至pET28a(+)中,将重组质粒pET28a-dsaDE转化至Escherichia coli BL21(DE3)中进行融合表达,利用Ni-NTA亲和层析法纯化融合蛋白DsaDE;分别以L-异亮氨酸和L-别异亮氨酸为底物进行融合蛋白的体外酶活性检测,利用高效液相色谱对酶反应产物进行分析。【结果】PCR验证、酶切验证以及测序结果证明pET28a-dsaDE重组载体具有正确序列;N-末端和C-末端融合6个组氨酸标签的融合蛋白DsaDE在E. coli BL21(DE3)中获得可溶性表达,经Ni-NTA亲和层析法一步纯化获得纯度约95%的融合蛋白,纯化的融合蛋白DsaDE具有较好的活性,能够催化L-isoleucine和L-allo-isoleucine间的相互转化。【结论】氨基转移酶DsaD和异构酶DsaE成功融合表达,经一步Ni-NTA亲和层析法纯化即可获得纯度较高的融合蛋白,融合蛋白同时具有氨基转移酶和异构酶的活性,为进一步研究L-别异亮氨酸的工业化生产奠定了基础。     

Motions and structural variability within toxins: implication for their use as scaffolds for protein engineering

Animal toxins are small proteins built on the basis of a few disulfide bonded frameworks. Because of their high variability in sequence and biologic function, these proteins are now used as templates for protein engineering. Here we report the extensive characterization of the structure and dynamics of two toxin folds, the "three-finger" fold and the short alpha/beta scorpion fold found in snake and scorpion venoms, respectively. These two folds have a very different architecture; the short alpha/beta scorpion fold is highly compact, whereas the "three-finger" fold is a beta structure presenting large flexible loops. First, the crystal structure of the snake toxin alpha was solved at 1.8-A resolution. Then, long molecular dynamics simulations (10 ns) in water boxes of the snake toxin alpha and the scorpion charybdotoxin were performed, starting either from the crystal or the solution structure. For both proteins, the crystal structure is stabilized by more hydrogen bonds than the solution structure, and the trajectory starting from the X-ray structure is more stable than the trajectory started from the NMR structure. The trajectories started from the X-ray structure are in agreement with the experimental NMR and X-ray data about the protein dynamics. Both proteins exhibit fast motions with an amplitude correlated to their secondary structure. In contrast, slower motions are essentially only observed in toxin alpha. The regions submitted to rare motions during the simulations are those that exhibit millisecond time-scale motions. Lastly, the structural variations within each fold family are described. The localization and the amplitude of these variations suggest that the regions presenting large-scale motions should be those tolerant to large insertions or deletions.     

HBcAg和HBsAg前S1表位肽融合蛋白的表达研究

构建、表达并纯化了HBcAg与HBsAg前S1表位肽融合蛋白BTcs1,为开发新型HBV治疗和预防性疫苗提供实验依据。利用DNA重组技术,构建了HBcAg与HBsAg前S1表位肽融合蛋白原核表达质粒pBTcs1,在大肠杆菌（HB101）中进行表达,用蔗糖密度梯度超速离心对表达产物BTcs1进行纯化,用SDS-PAGE、SEC、WESTERN-BLOT和电镜进行鉴定。结果表明成功构建了HBcAg与HBsAg前S1表位肽融合蛋白原核表达质粒pBTcs1,BTcs1的表达量为20-25 mg/L,DOT-BLOT结果显示BTcs1主要分布在30%-50%蔗糖介质中,SDS-PAGE和SEC结果显示蛋白纯度>95%,WESTERN-BLOT结果显示BTcs1可以与抗HBcAg抗体和抗HBsAg前S1抗体特异杂交,显色条带在约28 kD处,电镜分析表明BTcs1可以自主组装成病毒样颗粒（VLP）,直径约为30-34 nm。本研究为进一步探讨BTcs1的功能及应用奠定了基础。     

The construction and characterization of a bifunctional EGFP/sAPRIL fusion protein

A fusion protein of enhanced green fluorescent protein (EGFP) and soluble domain of human a proliferation-inducing ligand (sAPRIL) was efficiently expressed in Escherichia coli BL 21 (DE3). The soluble EGFP/sAPRIL, around 43 kDa, was purified in milligram amounts using metal chellate affinity chromatography and detected with anti-His₆ and anti-hsAPRIL monoclonal antibody. The chimeric protein exhibited similar fluorescence spectra with free EGFP. In vitro, purified EGFP/sAPRIL specifically bound receptor B cell maturation antigen (BCMA) detected by enzyme linked immunosorbent assay (ELISA) and receptors [including heparan sulfate proteoglycan (HSPGs)]-positive cell lines analyzed by fluorescence-activated cell sorting (FACS). Confocal laser microscopy images visibly showed the HSPGs’-dependent binding of EGFP/sAPRIL to NIH-3T3 cell. In addition, the chimera retained the bioactivity to stimulate/co-stimulate proliferation of NIH-3T3 and Jurkat cell/human B cell in vitro. Therefore, the fusion protein shows a readily obtainable source of biologically active sAPRIL which has considerable potential for single-step fluorescence detection assay in the study of APRIL and its receptors.     

The C-terminal ligand-binding domain of Clostridium difficile toxin A (TcdA) abrogates TcdA-specific binding to cells and prevents mouse lethality

We have investigated the ability of a recombinant protein (REP231), derived from Clostridium difficile toxin A C-terminal domain, to protect against toxin A (TcdA) intoxication in vitro and in vivo. REP231 was cloned, expressed and purified by thyroglobulin affinity chromatography, and demonstrated identical binding properties to TcdA. Immunofluorescence experiments and in vitro cytotoxicity assays using mouse teratocarcinoma cells F9 showed that specific binding of TcdA to F9 cells through its C-terminal domain is essential for producing cytotoxic effects. TcdA binding and cytotoxicity was inhibited by REP231 and a monoclonal antibody directed against the C-terminal domain. Toxin B did not bind to F9 cells and was consequently inactive in cytotoxicity assays. Inhibition studies with lectins and a Le^x-specific antibody supported earlier findings that a terminal galactose is part of the bound saccharide but excluded Le^x as a receptor for TcdA. Mice immunised with REP231 were protected against a threefold lethal dose of TcdA. Thus, REP231 appeared to be a suitable candidate to develop an alternative therapeutic agent, which is able to neutralise carbohydrate-mediated TcdA binding and might act as a vaccine.     

Removal of DnaK contamination during fusion protein purifications

The use of fusion proteins for recombinant protein expression in Escherichia coli has become popular because the carrier increases protein solubility, standardizes expression levels, and facilitates purification of the fusion products. However, we have observed that the peptide regions that fuse the carrier to the protein of interest bind E. coli Hsp70 molecular chaperones (DnaK) depending on their amino acid composition, resulting in an unwanted contamination during protein purification. Here we describe an approach that helps to circumvent this unwanted contamination. First, the appropriate amino acids surrounding and comprising the cloning site are chosen by using a software based on an algorithm already developed to decrease to a minimum the propensity of the fusion protein to bind DnaK. Second, DnaK contamination is significantly reduced by washing the fusion protein bound to the purification resin with MgATP plus soluble denatured E. coli proteins before elution. The approach can also be applied to eliminate other molecular chaperones.     

Synthesis of a disulfide-linked octameric peptide construct carrying three different antigenic determinants

In an effort to develop peptide vaccines against the influenza virus, we have successfully synthesized a disulfide-linked octameric homodimer that bears four copies of the influenza virus M2 protein ectodomain as well as two copies each of T-helper cell hemagglutinin epitopes, the I-E(d) restricted S1 and the I-A(d) restricted S2 fragments. Peptide attachment was via intermolecular disulfide formation from free sulfhydryl-bearing cysteine derivatives in solution. This reaction was efficient only when the amino-group of the cysteine was Fmoc-protected.     

Immobilisation of bovine enterokinase and application of the immobilised enzyme in fusion protein cleavage

Two immobilisation methods for enterokinase were developed, which yielded high remaining activities for the cleavage of the fusion protein MUC1-IgG Fc. Different carrier materials were compared regarding remaining enzyme activity and storage stability. Immobilisation procedures involving support material activation using glutardialdehyde were found to result in low remaining activities. Applying less aggressive activation procedures, remaining activities of approximately 60% were received when immobilising enterokinase on either Estapor paramagnetic microspheres or hexamethylamino Sepabeads. In case of hexamethylamino Sepabeads we were able to increase the half-life time 4.3-fold at 23 degrees C and 3.8-fold at 4 degrees C compared to the free enzyme at the same temperatures. By immobilising the biocatalyst the downstream process is simplified allowing the easy removal of the enzyme from the reaction mixture. The immobilised enterokinase cleaves the fusion protein MUC1-IgG Fc in at least two repeated batches, proving the efficiency of the immobilisation method and the reusability of the biocatalyst.