Maltose binding protein facilitates high-level expression and functional purification of the chemokines RANTES and SDF-1alpha from Escherichia coli

The chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and SDF-1α (stromal cell-derived factor-1α) are important regulators of leukocyte trafficking and homing. Chemokines form insoluble inclusion bodies when expressed in Escherichia coli (E. coli), resulting in low yields of soluble protein. We have developed a novel chemokine expression system that generates a high amount of soluble protein and uses a simple purification scheme. We cloned different types of RANTES and SDF-1α fused to either maltose binding protein (MBP) or glutathione-S-transferase (GST) and expressed the fusion proteins in E. coli under various conditions. We found that the yield of soluble chemokine is influenced by the type of fusion partner. Fusion to MBP resulted in a higher yield of total and soluble chemokine compared to GST. Under optimized conditions, the yield of soluble MBP–RANTES and MBP–SDF-1α was 2.5- and 4.5-fold higher than that of the corresponding GST-fusion protein, respectively. Recombinant chemokine fusion proteins exhibited specific binding activity to chemokine receptors. These results demonstrate that the use of MBP-fusion proteins may provide an approach to generating high yields of soluble and functional chemokines, such as RANTES and SDF-1α.     

密码子优化型HPV16L1基因在两种昆虫细胞中的表达

目的:提高16型人乳头瘤病毒（HPV16）L1基因在杆状病毒昆虫细胞中的表达水平,为研制预防性HPV疫苗奠定基础。方法:根据昆虫细胞密码子偏性对野生型HPV16L1基因进行改造,利用Bac-to-Bac表达系统获得重组杆状病毒,感染昆虫细胞Sf9和High Five。Western blot鉴定表达产物;电镜下观察病毒样颗粒形成。利用ELISA法评价HPV16L1基因的优化效果,探讨L1蛋白表达的最佳条件。结果:在相对分子质量56kDa处出现HPV16L1的特异性条带;电镜下可见病毒样颗粒在昆虫细胞的核内形成;优化型HPV16L1基因的表达水平显著高于野生型。High Five细胞表达的最佳条件为MOI=10,表达时相72h,其L1蛋白表达量至少比Sf9细胞高3倍。结论:密码子优化技术确实能够促进HPV16L1蛋白的高效表达,而High Five细胞表现出的显著优势尤其值得关注。     

Escherichia coli maltose-binding protein is uncommonly effective at promoting the solubility of polypeptides to which it is fused.

Although it is usually possible to achieve a favorable yield of a recombinant protein in Escherichia coli, obtaining the protein in a soluble, biologically active form continues to be a major challenge. Sometimes this problem can be overcome by fusing an aggregation-prone polypeptide to a highly soluble partner. To study this phenomenon in greater detail, we compared the ability of three soluble fusion partners--maltose-binding protein (MBP), glutathione S-transferase (GST), and thioredoxin (TRX)--to inhibit the aggregation of six diverse proteins that normally accumulate in an insoluble form. Remarkably, we found that MBP is a far more effective solubilizing agent than the other two fusion partners. Moreover, we demonstrated that in some cases fusion to MBP can promote the proper folding of the attached protein into its biologically active conformation. Thus, MBP seems to be capable of functioning as a general molecular chaperone in the context of a fusion protein. A model is proposed to explain how MBP promotes the solubility and influences the folding of its fusion partners.     

利用重组HPV16L1抗原检测宫颈癌抗L1或VLP抗体的对比

为了评价重组大肠杆菌表达的HPV16L1蛋白和重组腺病毒表达的HPV16L1 VLP两种抗原在检测宫颈癌抗 16L1或VLP抗体及在宫颈癌血清学诊断意义上的差别 ,应用PCR技术从宫颈癌组织的DNA中扩增出全长15 35bp的HPV16L1基因片段 ,克隆至 pUC18 T载体中 ,进行DNA测序鉴定。然后 ,将HPV16L1基因克隆至pGEX 2T表达载体中 ,并诱导表达HPV16L1融合蛋白 ,分子量为 83kD ,能被HPV16L1单克隆抗体所识别。经GST柱层析法纯化后 ,与重组腺病毒表达的HPV16L1 VLP分别经酶联免疫吸附 (ELISA)法检测 12份宫颈癌患者和 35份献血员血清。 12例宫颈癌血清标本中 ,抗HPV16L1蛋白的抗体阳性率为 7例 (占 5 8.3% ) ;抗HPV16L1 VLP的抗体阳性率为 8例 (占 6 6 .7% )。经大肠杆菌表达的重组抗原HPV16L1检测为HPV16抗体IgG( )的 7份患者血清 ,利用HPV16L1 VLP试剂盒检测均阳性 ;经大肠杆菌表达的重组抗原检测为HPV16抗体IgG( )的 5份患者血清 ,利用HPV16L1 VLP试剂盒检测有 1份阳性。两者对HPV16抗体的阳性检出率并无显著差异 (P >0 .0 5 )。本实验结果说明HPV16与宫颈癌高度相关 ,利用大肠杆菌表达的重组抗原HPV16L1和HPV16L1 VLP重组抗原检测抗体的敏感性并不受影响。利用重组抗原HPV16L1对宫颈癌的抗体进行定性、定量分析有助于该疾病     

Soluble expression,purification, and characterization of recombinant human flotillin-2 (reggie-1) in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Large scale production of recombinant human flotillin-2 (reggie-1) is desirable for structural and biochemical studies. However, as the major lipid rafts specific hydrophobic protein, flotillin-2 was difficult to be expressed as soluble and functional form in prokaryotic system. In this study, we first cloned and expressed human flotillin-2 in Escherichia coli with five different fusion tags: poly-histidine, glutathione S-transferase (GST), thioredoxin (TRX), N-Utilization substance (NusA) and maltose binding protein (MBP). We screened the expression level and solubility of the five flotillin-2 fusion proteins, the best MBP tagged flotillin-2 was then large scale produced. The optimized purification procedure included two steps of chromatography: Ni-NTA affinity chromatography and anion exchange chromatography. The typical yield was 36.0 mg soluble and functional recombinant flotillin-2 from 1 L of culture medium with purity above 97%. The activity of recombinant flotillin-2 was verified by pull-down assay with flotillin-1, showing that the purified recombinant flotillin-2 can specifically interact with flotillin-1. The circular dichroism (CD) spectroscopy showed that recombinant flotillin-2 had a very stable secondary structure dominated by α-helix, β-turn and random structure.     

Bacterial expression and purification of human papillomavirus type 18 L1

The human papillomavirus (HPV) 18 L1 gene, which encodes the L1 major capsid protein, was isolated from a female patient in Pusan, Korea Republic and was cloned into pGEX-4T-1 vector. The HPV-18L1 gene was expressed in Escherichia coli as a fusion protein with a glutathione-S-transferase (GST) tag. The soluble recombinant fusion protein, GST-18 L1 fusion, was isolated to high purity. HPV-18 L1 was purified from the GST-18 L1 fusant after biotinylated thrombin cleavage, and then the treated thrombin was removed serially using streptavidin conjugated resin. The purified HPV-18 L1 was confirmed by western blotting using a rabbit anti-denatured papillomavirus polyclonal antibody. The virus-like particles (VLP) from the purified full-length 18 L1 protein without any extra amino acid sequences was observed through the analysis of the electron microscope. This is the first study to report the expression and purification of HPV-18 L1 in E. coli. This expression and purification system offers a simple method of expressing and purifying HPV L1 protein, and could potentially be an effective route for the development and manufacturing of highly purified HPV-18 L1-based cervical cancer vaccines.     

人乳头瘤病毒16型E7蛋白的原核表达与鉴定

本研究在大肠杆菌BL21中融合表达人乳头瘤病毒16型E7蛋白,并初步评价其应用价值。采用PCR技术扩增出HPV16E7基因,将其克隆进原核表达载体pGEX6p-1,转化至大肠杆菌BL21,利用IPTG进行诱导表达。以纯化的融合蛋白作为检测抗原建立间接ELISA方法,用于检测重组李斯特菌(Lm1-2-E7)免疫小鼠后的E7血清抗体水平。在25℃,0.5mM IPTG诱导下,HPV16E7蛋白在大肠杆菌BL21中获得表达,融合蛋白以可溶性形式存在,Western blot结果显示其与HPV16E7单克隆抗体发生特异性反应。二次免疫后小鼠血清经间接ELISA结果表明E7特异性抗体滴度为1∶200。结果表明GST-E7融合蛋白具有较强的免疫活性。     

Expression of membrane proteins from Mycobacterium tuberculosis in Escherichia coli as fusions with maltose binding protein

Sixteen of 22 low molecular weight integral membrane proteins from Mycobacterium tuberculosis with previously poor or undetectable levels of expression were expressed in Escherichia coli as fusions with both the maltose binding protein (MBP) and a His(8)-tag. Sixty-eight percent of targeted proteins were expressed in high yield (>30 mg/L) in soluble and/or inclusion body form. Thrombin cleavage of the MBP fusion protein was successful for 10 of 13 proteins expressed as soluble proteins and for three proteins expressed only as inclusion bodies. The use of autoinduction growth media increased yields over Luria-Bertani (LB) growth media in 75% of the expressed proteins. Expressing integral membrane proteins with yields suitable for structural studies from a set of previously low and non-expressing proteins proved highly successful upon attachment of the maltose binding protein as a fusion tag.     

Protein mutagenesis with monodispersity-based quality probing: selective inactivation of p53 degradation and DNA-binding properties of HPV E6 oncoprotein

Interpretation of protein mutagenesis experiments requires the ability to distinguish functionally relevant mutations from mutations affecting the structure. When a protein is expressed soluble in bacteria, properly folded mutants are expected to remain soluble whereas misfolded mutants should form insoluble aggregates. However, this rule may fail for proteins fused to highly soluble carrier proteins. In a previous study, we analysed the biophysical status of HPV oncoprotein E6 fused to the C-terminus of maltose-binding protein (MBP) and found that misfolded E6 moieties fused to MBP formed soluble aggregates of high molecular weight. By contrast, preparations of properly folded E6 fused to MBP were monodisperse. Here, we have used this finding to evaluate the quality of 19 MBP-fused E6 site-directed mutants by using a light scattering assay performed in a fluorimeter. This assay guided us to rule out structurally defective mutants and to obtain functionally relevant E6 mutants selectively altered for two molecular activities: degradation of tumour suppressor p53 and DNA recognition.     

人乳头瘤病毒18型L1蛋白的包涵体和可溶性表达及纯化

目的:原核表达系统表达人乳头瘤病毒18型(HPV18)L1蛋白,建立包涵体和可溶性表达的L1蛋白的纯化方法。方法:构建重组表达质粒p GEX-4T-1-HPV18 L1,在大肠杆菌BL21中以包涵体和可溶性方式表达HPV18 L1蛋白。通过超声波破碎菌体、洗涤包涵体、碱变性、透析复性和谷胱甘肽(GST)琼脂糖凝胶4B亲和层析纯化包涵体蛋白;在菌体中加入三磷酸腺苷(ATP)和3.5 mol/L尿素孵育后,GST 4B亲和层析纯化可溶性蛋白,凝血酶酶切。SDS-PAGE和Western印迹鉴定表达和纯化产物。结果:SDS-PAGE结果表明,HPV18 L1蛋白以包涵体和可溶性方式在大肠杆菌BL21内高效表达,均产生相对分子质量约为86 000的HPV18 L1-GST融合蛋白。Western印迹结果显示,包涵体纯化后获得的融合蛋白降解条带较多;而可溶性蛋白纯化后获得的融合蛋白未降解,凝血酶酶切后得到HPV18 L1蛋白,可与HPV18 L1蛋白单克隆抗体结合。结论:采用原核系统表达了HPV18 L1-GST融合蛋白,分别建立了包涵体和可溶性蛋白的纯化方法,获得HPV18 L1蛋白,为其进一步应用奠定了基础。     

Plastid expression of a double-pentameric vaccine candidate containing human papillomavirus-16 L1 antigen fused with LTB as adjuvant: transplastomic plants show pleiotropic phenotypes

Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus-like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the highest burden of disease in resource-poor countries, development of an improved mucosal and cost-effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate. Furthermore, coupling of an adjuvant like Escherichia coli heat-labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co-administration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV-16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB-L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1-ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild-type plants. Taken together, these results pave the way for the possible development of a low-cost adjuvant-coupled vaccine with potentially improved immunogenicity against cervical cancer.     

Human papillomavirus L1 protein expressed in tobacco chloroplasts self-assembles into virus-like particles that are highly immunogenic

Cervical cancer is the second most prevalent cancer in women worldwide. It is linked to infection with human papillomavirus (HPV). As the virus cannot be propagated in culture, vaccines based on virus-like particles have been developed and recently marketed. However, their high costs constitute an important drawback for widespread use in developing countries, where the incidence of cervical cancer is highest. In a search for alternative production systems, the major structural protein of the HPV-16 capsid, L1, was expressed in tobacco chloroplasts. A very high yield of production was achieved in mature plants (approximately 3 mg L1/g fresh weight; equivalent to 24% of total soluble protein). This is the highest expression level of HPV L1 protein reported in plants. A single mature plant synthesized approximately 240 mg of L1. The chloroplast-derived L1 protein displayed conformation-specific epitopes and assembled into virus-like particles, visible by transmission electron microscopy. Furthermore, leaf protein extracts from L1 transgenic plants were highly immunogenic in mice after intraperitoneal injection, and neutralizing antibodies were detected. Taken together, these results predict a promising future for the development of a plant-based vaccine against HPV.     

Expression and purification of receptor for activated C-kinase 1 (RACK1)

Receptor for activated C-kinase (RACK1) binds to protein kinase C and functions as an anchor for several other cellular components. Most in vitro studies of RACK1 have been carried out with RACK1 fused to a soluble fusion protein partner, such as GST or MBP. Here, we show that fusion complexes may exist as large soluble aggregates and thereby lead to false conclusions about the biological activity of RACK1. We developed a purification procedure that gave soluble monodisperse molecules of the protein. The RACK1 gene was cloned and expressed in a pMAL vector. After purification of the resulting MBP-RACK1 fusion protein, RACK1 was excised from MBP by thrombin, rendering RACK1 in a soluble monodisperse form as monitored by fluorimetric static light scattering, gel filtration, and ultracentrifugation. Circular dichroism analysis revealed that RACK1 was properly folded with a T(m) of approximately 62 degrees C and contained the predicted portions of secondary structures. The biological activity of the purified protein was verified by binding to activated protein kinase C. The production of soluble, high-purity RACK1 will allow structural studies and functional in vitro studies to identify interacting partners to this important scaffold protein.     

Characterization of factors favoring the expression of soluble protozoan tubulin proteins in Escherichia coli

The alpha- and beta-tubulin genes of the parasitic protozoa Giardia duodenalis, Cryptosporidium parvum, and Encephalitozoon intestinalis have been overexpressed in soluble form using Escherichia coli-based expression systems. Several expression systems were compared in terms of the amount of soluble protein produced with different fusion partners, strains of E. coli BL21, and expression temperatures. The cleavability of the fusion partner was also assessed in terms of post-expression applications of the recombinant protein. The maltose-binding protein (MBP) and glutathione S-transferase (GST) fusion partners produced the highest expression levels for all six proteins without the formation of inclusion bodies. The expression system also provided a means of purifying the soluble protein using affinity and anion-exchange chromatography while minimizing protein losses. The yield and purity were therefore very high for both the MBP and GST systems. The tubulin monomers were demonstrated to be assembly-competent using a standard dimerization assay and also retained full antigenicity with monoclonal antibodies. This study presents several methods which are suitable for producing soluble tubulin monomers and, thus, circumventing the formation of inclusion bodies which necessitates re-folding of the tubulin.     

Expression of human papillomavirus type 16 L1 protein in transgenic tobacco plants

To develop a plant expression system for the production of the human papillomavirus type 16 (HPV16) vaccine, we investigated whether the HPV16 L1 protein can be expressed in tobacco plants and whether it can be used as the cheapest form of edible vaccine. The HPV16 L1 coding sequence was amplified by PCR using specific primers from the plasmid pGEM-T-HPV16 containing the template sequence, and subcloned into the intermediate vector pUCmT and binary vector pBI121 consecutively to obtain the plant expression plasmid pBI-L1. The T-DNA regions of the pBI-L1 binary vector contained the constitutive Cauliflower mosaic virus (CaMV) 35S promoter and the neomycin phosphotransferase npt Ⅱ gene, which allowed the selection of transformed plants using kanamycin. The tobacco plants were transformed by cocultivating them, using the leaf disc method, with Agrobacterium tumefaciens LBA4404, which harbored the plant expression plasmid. The regenerated transgenic tobacco plants were selected using kanamycin, and confirmed by PCR. The results of the Southern blot assay also showed that the HPV16 L1 gene was integrated stably into the genome of the transformed tobacco plants. The Western blot analysis showed that the transformed tobacco leaves could express the HPV 16 L1 protein. Furthermore, it was demonstrated by ELISA assay that the expressed protein accounted for 0.034%-0.076% of the total soluble leaf protein, was able to form 55nm virus-like particles compatible with HPV virus-like particle (VLP), and induced mouse erythrocyte hemagglutination in vitro. The present results indicate that the HPV 16 L1 protein can be expressed in transgenic tobacco plants and the expressed protein possesses the natural features of the HPV16 L1 protein, implying that the HPV16 L1 transgenic plants can be potentially used as an edible vaccine.     

Characterization of HPV16 L1 loop domains in the formation of a type-specific,conformational epitope

Background  

Virus-like particles (VLPs) formed by the human papillomavirus (HPV) L1 capsid protein are currently being tested in clinical trials as prophylactic vaccines against genital warts and cervical cancer. The efficacy of these vaccines is critically dependent upon L1 type-specific conformational epitopes. To investigate the molecular determinants of the HPV16 L1 conformational epitope recognized by monoclonal antibody 16A, we utilized a domain-swapping approach to generate a series of L1 proteins composed of a canine oral papillomavirus (COPV) L1 backbone containing different regions of HPV16 L1.     

Transplastomic expression of a modified human papillomavirus L1 protein leading to the assembly of capsomeres in tobacco: a step towards cost-effective second-generation vaccines

Certain types of human papillomaviruses (HPV) are causatively associated with cervical carcinoma, the second most common cancer in women worldwide. Due to limitations in the availability of currently used virus-like particle (VLP)-based vaccines against HPV to women of developing countries, where most cases of cervical cancer occur, the development of a cost-effective second-generation vaccine is a necessity. Capsomeres have recently been demonstrated to be highly immunogenic and to have a number of advantages as a potential cost-effective alternative to VLP-based HPV vaccines. We have expressed a mutated HPV-16 L1 (L1_2xCysM) gene that retained the ability to assemble L1 protein to capsomeres in tobacco chloroplasts. The recombinant protein yielded up to 1.5% of total soluble protein. The assembly of capsomeres was examined and verified by cesium chloride density gradient centrifugation and sucrose sedimentation analysis. An antigen capture enzyme-linked immunosorbent assay confirmed the formation of capsomeres by using a conformation-specific monoclonal antibody which recognized the conformational epitopes. Transplastomic tobacco plants exhibited normal growth and morphology, but all such lines showed male sterility in the T₀, T₁ and T₂ generations. Taken together, these results indicate the possibility of producing a low-cost capsomere-based vaccine by plastids.     

Formation of soluble inclusion bodies by hpv e6 oncoprotein fused to maltose-binding protein

Many polypeptides overexpressed in bacteria are produced misfolded and accumulate as solid structures called inclusion bodies. Inclusion-body-prone proteins have often been reported to escape precipitation when fused to maltose-binding protein (MBP). Here, we have examined the case of HPV 16 oncoprotein E6. The unfused sequence of E6 is overexpressed as inclusion bodies in bacteria. By contrast, fusions of E6 to the C-terminus of MBP are produced soluble. We have analyzed preparations of soluble MBP-E6 fusions by using three independent approaches: dynamic light scattering, lateral turbidimetry, and sandwich ELISA. All three methods showed that MBP-E6 preparations contain highly aggregated material. The behavior of these soluble aggregates under denaturating conditions suggests that they are formed by agglomeration of misfolded E6 moieties. However, precipitation is prevented by the presence of the folded and highly soluble MBP moieties, which maintain the aggregates in solution. Therefore, the fact that a protein or protein domain is produced soluble when fused to the C-terminus of a carrier protein does not guarantee that the protein of interest is properly folded and active. We suggest that aggregation of fusion proteins should be systematically assayed, especially when these fusions are to be used for binding measurements or activity tests.     

粗提和纯化的HPV16L1重组蛋白为抗原的ELISA比较

为评价真核及原核细胞表达的HPV16L1粗提蛋白代替纯化的16L1蛋白作为ELISA检测抗原的可行性。实验分别用大肠埃希菌表达的HPV16L1纯化蛋白,表达16L1的大肠埃希菌破碎物及表达16L1的昆虫细胞破碎物作为抗原。在ELISA试验中与抗HPV16L1的单克隆抗体进行反应。结果证实3种包被抗原可以得出非常相近的OD值变化趋势。表明用表达16L1的大肠埃希菌破碎物和表达16L1的昆虫细胞破碎物可以代替纯化的HPV16L1蛋白作为抗原进行ELISA试验。     

Evaluation of serum antibody response to a newly identified B-cell epitope in the minor nucleocapsid protein L2 of human papillomavirus type 16

The aim of this work was to identify B-cell epitopes in the minor nucleocapsid (L2) protein of human papillomavirus (HPV) type 16 and characterization of allied antibody response. Serum samples of 513 individuals (323 women with various degrees of cervical atypia, 150 men and 40 small children) were available for the study. Synthetic peptides overlapping the L2 protein of HPV 16 twice were applied in ELISA for epitope scanning and antibody determination. An HPV 16 L2 derived dodecamer SGYIPANTTIPF (amino acids 391-402) proved to be the major B-cell epitope. Both IgA antipeptide antibody positivity (range 7-28%) and mean IgA antibody levels (range 13.2 EIU to 42.4 EIU, P < 0.05) increased with the degree of cervical atypia, whereas antipeptide IgG antibodies showed an opposite trend. During a 2-years follow-up significantly (P < 0.0005) decreasing IgA antibody levels to the SGYIPANTTIPF peptide were associated with regression of koilocytotic atypia. Analysis of anti-peptide IgA antibodies of 118 women with known HPV type revealed that a majority of positives had HPV 16/18 DNA. It was concluded that antibody response to the newly discovered peptide was partially type- and disease-specific. Our results also suggest an impairment of the IgG but not IgA class antibody response to HPV 16 in patients with persistent cervical HPV infection.