Visualisation of plastids in endosperm,pollen and roots of transgenic wheat expressing modified GFP fused to transit peptides from wheat SSU RubisCO,rice FtsZ and maize ferredoxin III proteins

The ability to target marker proteins to specific subcellular compartments is a powerful research tool to study the structure and development of organelles. Here transit sequences from nuclear-encoded, plastid proteins, namely rice FtsZ, maize non-photosynthetic ferredoxin III (FdIII) and the small subunit of RubisCO were used to target a modified synthetic GFP (S65G, S72A) to plastids. The localisations of the fusion proteins expressed in transgenic wheat plants and under the control of the rice actin promoter were compared to an untargeted GFP control. GFP fluorescence was localised to non-green plastids in pollen, roots and seed endosperm and detected in isolated leaf chloroplasts using a GFP-specific antibody. Transit peptides appeared to influence the relative fluorescence intensities of plastids in different tissues. This is consistent with differential targeting and/or turnover of GFP fusion proteins in different plastid types. Replacement of GFP sequences with alternative coding regions enables immediate applications of our vectors for academic research and commercial applications.     

An engineered chaperonin caging a guest protein: Structural insights and potential as a protein expression tool

The structure of a chaperonin caging a substrate protein is not quite clear. We made engineered group II chaperonins fused with a guest protein and analyzed their structural and functional features. Thermococcus sp. KS-1 chaperonin alpha-subunit (TCP) which forms an eightfold symmetric double-ring structure was used. Expression plasmids were constructed which carried two or four TCP genes ligated head to tail in phase and a target protein gene at the 3' end of the linked TCP genes. Electron microscopy showed that the expressed gene products with the molecular sizes of ~120 kDa (di-TCP) and ~230 kDa (tetra-TCP) formed double-ring complexes similar to those of wild-type TCP. The tetra-TCP retained ATPase activity and its thermostability was significantly higher than that of the wild type. A 260-kDa fusion protein of tetra-TCP and green fluorescent protein (GFP, 27 kDa) was able to form the double-ring complexes with green fluorescence. Image analyses indicated that the GFP moiety of tetra-TCP/GFP fusion protein was accommodated in the central cavity, and tetra-TCP/GFP formed the closed-form similar to that crystallographically resolved in group II chaperonins. Furthermore, it was suggested that caging GFP expanded the cavity around the bottom. Using this tetra-TCP fusion strategy, two virus structural proteins (21-25 kDa) toxic to host cells or two antibody fragments (25-36 kDa) prone to aggregate were well expressed in the soluble fraction of Escherichia coli. These fusion products also assembled to double-ring complexes, suggesting encapsulation of the guest proteins. The antibody fragments liberated by site-specific protease digestion exhibited ligand-binding activities.     

Regional assignment of the genes for TK1, GALK, ALDC, and ESD on chromosome 8 in the American mink by chromosome-mediated gene transfer

Summary A panel of clones of mink-Chinese hamster somatic cell hybrids was analysed to obtain data for assigning the genes for thymidine kinase-1 (TK1), galactokinase (GALK), subunit C of aldolase (ALDC), and esterase D (ESD) to specific mink chromosomes. The results demonstrate that the genes for TK1, GALK, ALDC and ESD are syntenic and located on mink chromosome 8. Prometaphase analysis of transformed mouse cells obtained by transfer of mink genes by means of metaphase chromosomes demonstrated the presence of mink chromosome 8 fragments of different sizes in some of the independent transformants. Segregation analysis of these fragments and mink TK1, GALK, ALDC and ESD allowed us to assign the genes for TK1 and GALK to 8p24, ALDC to pter-8p25, and ESD to 8q24-8qter.     

GST-HAI-1融合蛋白的表达及抗人HAI-1单克隆抗体的制备

制备抗人肝细胞生长因子激活物抑制因子（HAI-1）单克隆抗体,为对HAI-1进行进一步的研究打下基础。将人HAI-1 cDNA分段克隆,构建GST-HAI-1融合蛋白原核表达载体,转化大肠杆菌后加IPTG诱导融合蛋白表达,经制备型SDS-PAGE法分离表达的GST-HAI-1融合蛋白,通过割胶、电洗脱回收融合蛋白,并以此为抗原免疫BALB/c小鼠,应用细胞融合技术制备产生抗人HAI-1单克隆抗体的杂交瘤,以ELISA、Western blot和免疫组织化学染色进行鉴定。最终获得抗人HAI-1单克隆抗体杂交瘤细胞株ZMC6,产生的单克隆抗体可特异性地与表达的GST-HAI-1融合蛋白反应,并可识别大肠组织中的膜型及脱落型HAI-1蛋白。该单克隆抗体的制备成功,为深入研究HAI-1的功能提供了有力工具。     

Single-chain antibody against human lipocalin-type prostaglandin D synthase: Construction, expression, purification, and activity assay

An active form of single-chain antibody (ScFv) from murine monoclonal antibody 4A7, which is specific for lipocalin-type prostaglandin D synthase (L-PGDS), was produced in Escherichia coli. The complementary DNA fragments encoding the variable regions of heavy chain (VH) and light chain (VL), which amplified from hybridoma 4A7 producing a monoclonal antibody (IgG1) against L-PGDS, were connected by a (Gly4Ser)3 linker using an assembly polymerase chain reaction. The resultant ScFv were cloned into the vector pGEM and expressed in E. coli as inclusion bodies. The expressed ScFv fusion proteins were purified by Ni2+-nitrilotriacetic acid chromatography. The purity and activity of purified ScFv were confirmed by SDS-PAGE and ELISA. The result revealed that 4A7 ScFv conserved the same characteristics of specific recognition and binding to sperm as the parental 4A7 monoclonal antibody.     

Generation of monoclonal antibodies for the assessment of protein purification by recombinant ribosomal coupling

We recently described a conceptually novel method for the purification of recombinant proteins with a propensity to form inclusion bodies in the cytoplasm of Escherichia coli. Recombinant proteins were covalently coupled to the E. coli ribosome by fusing them to ribosomal protein 23 (rpL23) followed by expression in an rpL23 deficient strain of E. coli. This allowed for the isolation of ribsomes with covalently coupled target proteins which could be efficiently purified by centrifugation after in vitro proteolysis at a specific site incorporated between rpL23 and the target protein. rpL23-GFP-His is among the fusion proteins used in our previous study for ribosomal coupling of C-terminally His-tagged green fluorescent protein. To assess the efficiency of separation of target protein from ribosomes, by site-specific proteolysis, we required monoclonal antibodies directed against rpL23 and GFP. We therefore purified rpL23-GFP-His, rpL23-His and GFP from E. coli recombinants using affinity, ion exchange and hydrophobic interaction chromatography. These proteins could be purified with yields of 150, 150 and 1500 microg per gram cellular wet weight, respectively. However, rpL23-GFP-His could only be expressed in a soluble form and subsequently purified, when cells were cultivated at reduced temperatures. The purified rpL23-GFP-His fusion protein was used to immunize balb/c mice and the hybridoma cell lines resulting from in vitro cell fusion were screened by ELISA using rpL23-His and GFP to select for monoclonal antibodies specific for each protein. This resulted in 20 antibodies directed against rpL23 and 3 antibodies directed against GFP. Antibodies were screened for isotypes and their efficiency in western immunoblots. The most efficient antibody against rpL23 and GFP were purified by Protein G Sepharose affinity chromatography. The purified antibodies were used to evaluate the separation of ribosomes from GFP, streptavidin, murine interleukin-6, a phagedisplay antibody and yeast elongation factor 1A by centrifugation, when ribosomes with covalently coupled target protein were cleaved at specific proteolytic cleavage sites. We conclude that the generated antibodies can be used to evaluate ribosomal coupling of recombinant target proteins as well as the efficiency of their separation from the ribosome.     

Improved quantitative and qualitative production of single-domain intrabodies mediated by the co-expression of Erv1p sulfhydryl oxidase

Camelidae single domain antibodies (VHHs) have structural and binding features that render them suitable alternatives to conventional IgG antibodies. VHHs are usually easier to produce as recombinant proteins than other antibody fragments. However, for some of the biotechnological applications for which they have been proposed, such as immunochromatography and assisted-crystallography, large amounts of purified antibodies are necessary, whereas some VHH-fusions with common tags such as GFP and SNAP are poorly expressed in the bacterial periplasm. Here we have shown that the co-expression of Erv1p sulfhydryl oxidase resulted in an astonishing yield increase of VHH-SNAP constructs expressed in the bacterial cytoplasm. The resulting recombinant antibodies were also more stable than the antibodies produced using the same plasmid, but in wild-type bacteria. Using this approach, it was possible to obtain tens of milligram of purified fusion antibodies using a basic flask fermentation protocol. Therefore, the described method represents a valid solution to produce inexpensively large amounts of single domain antibodies for in vitro applications and we expect it will be suitable for the production of other antibody fragments.     

The identification of Mycobacterium marinum genes differentially expressed in macrophage phagosomes using promoter fusions to green fluorescent protein

Mycobacterium marinum , like Mycobacterium tuberculosis , is a slow-growing pathogenic mycobacteria that is able to survive and replicate in macrophages. Using the promoter-capture vector pFPV27, we have constructed a library of 200–1000 bp fragments of M. marinum genomic DNA inserted upstream of a promoterless green fluorescent protein (GFP) gene. Only those plasmids that contain an active promoter will express GFP. Macrophages were infected with this fusion library, and phagosomes containing fluorescent bacteria were isolated. Promoter constructs that were more active intracellularly were isolated with a fluorescence-activated cell sorter, and inserts were partially sequenced. The promoter fusions expressed intracellularly exhibited homology to mycobacterial genes encoding, among others, membrane proteins and biosynthetic enzymes. Intracellular expression of GFP was 2–20 times that of the same clones grown in media. Several promoter constructs were transformed into Mycobacterium smegmatis , Mycobacterium bovis BCG and Mycobacterium tuberculosis . These constructs were positive for GFP expression in all mycobacterial strains tested. Sorting fluorescent bacteria in phagosomes circumvents the problem of isolating a single clone from macrophages, which may contain a mixed bacterial population. This method has enabled us to isolate 12 M. marinum clones that contain promoter constructs differentially expressed in the macrophage.     

Production of fluorescent single-chain antibody fragments in Escherichia coli

We describe a novel vector-host system suitable for the efficient preparation of fluorescent single-chain antibody Fv fragments (scFv) in Escherichia coli. The previously described pscFv1F4 vector used for the bacterial expression of functional scFv to the E6 protein of human papillomavirus type 16 was modified by appending to its C-terminus the green fluorescent protein (GFP). The expression of the scFv1F4-GFP fusion proteins was monitored by analyzing of the typical GFP fluorescence of the transformed cells under UV illumination. The brightest signal was obtained when scFv1F4 was linked to the cycle 3 GFP variant (GFPuv) and expressed in the cytoplasm of AD494(DE3) bacteria under control of the arabinose promoter. Although the scFv1F4 expressed under these conditions did not contain disulfide bridges, about 1% of the molecules were able to bind antigen. Fluorescence analysis of antigen-coated agarose beads incubated with the cytoplasmic scFv-GFP complexes showed that a similar proportion of fusions retained both E6-binding and green-light-emitting activities. The scFv1F4-GFPuv molecules were purified by affinity chromatography and successfully used to detect viral E6 protein in transfected COS cells by fluorescence microscopy. When an anti-beta-galactosidase scFv, which had previously been adapted to cytoplasmic expression at high levels, was used in this system, it was possible to produce large amounts of functional fluorescent antibody fragments. This indicates that these labeled scFvs may have many applications in fluorescence-based single-step immunoassays.     

Green fluorescent protein (GFP) expression patterns in the olfactory epithelium of GFP transgenic cloned Jinhua pigs

Domestic pigs possess a well‐developed sense of smell. However, the morphology of the porcine olfactory epithelium (OE) is poorly understood. Recently, several strains of transgenic cloned pigs that are presumed to ubiquitously express green fluorescent protein (GFP) have been created. Thus, the purpose of this study was to elucidate the features of porcine OE using the tissues of GFP transgenic cloned pigs. Based on observations of Hematoxylin and Eosin staining and measurements of thickness, porcine OE tissue portions were classified into three categories (thick, standard, and thin). Cryosections revealed that the prominent GFP signals were expressed in olfactory sensory neurons (OSN), Bowman's glands, and olfactory nerve. A few GFP‐expressing sustentacular cells were seen; however, the intensity of GFP fluorescence was slight. In the thick portion, numerous GFP‐expressing polygonal OSN that did not possess dendrites were found. In the standard portions, GFP‐expressing cells had longitudinal dendrites. A few GFP‐expressing cells were found in the thin portion. In the thick and standard portions, most of the prominent GFP‐expressing cells were positive for olfactory marker protein. Moreover, double immunofluorescence staining with boiled GFP and Sox2 antibody revealed that GFP expression patterns in OSN are synchronized with Sox2 immunoreactive patterns.     

In vivo and in vitro protein solubility assays using split GFP

The rapid assessment of protein solubility is essential for evaluating expressed proteins and protein variants for use as reagents for downstream studies. Solubility screens based on antibody blots are complex and have limited screening capacity. Protein solubility screens using split beta-galactosidase in vivo and in vitro can perturb protein folding. Split GFP used for monitoring protein interactions folds poorly, and to overcome this limitation, we recently developed a protein-tagging system based on self-complementing split GFP derived from an exceptionally well folded variant of GFP termed 'superfolder GFP'. Here we present the step-by-step procedure of the solubility assay using split GFP. A 15-amino-acid GFP fragment, GFP 11, is fused to a test protein. The GFP 1-10 detector fragment is expressed separately. These fragments associate spontaneously to form fluorescent GFP. The fragments are soluble, and the GFP 11 tag has minimal effect on protein solubility and folding. We describe high-throughput protein solubility screens amenable both for in vivo and in vitro formats. The split-GFP system is composed of two vectors used in the same strain: pTET GFP 11 and pET GFP 1-10 (Fig. 1 and Supplementary Note online). The gene encoding the protein of interest is cloned into the pTET GFP 11 vector (resulting in an N-terminal fusion) and transformed into Escherichia coli BL21 (DE3) cells containing the pET GFP 1-10 plasmid. We also describe how this system can be used for selecting soluble proteins from a library of variants (Box 1). The large screening power of the in vivo assay combined with the high accuracy of the in vitro assay point to the efficiency of this two-step split-GFP tool for identifying soluble clones suitable for purification and downstream applications.     

A human kringle domain-based fluorescence-linked immunosorbent assay system

As a non-immunoglobulin protein scaffold, human kringle domain (KD) has attractive properties such as high specificity, stability, and production in bacterial hosts. Here, we developed a rapid and sensitive fluorescence-linked immunosorbent assay (FLISA) system using a fluorescent kringle domain (fluoKD), a fusion protein of a green fluorescent protein (GFP), and a kringle domain variant (KD548). Two kinds of fluoKDs in which KD was fused to the N terminus of GFP (N-fluoKD) or the C terminus of GFP (C-fluoKD) were constructed and characterized. In Escherichia coli host, both fluoKDs were produced in high yield and solubility and were successfully purified by a simple procedure. The purified fluoKDs exhibited strong fluorescent activities and high affinities to the target antigen. Furthermore, it was successfully demonstrated that the FLISA with purified fluoKDs allowed for more rapid detection of target antigens with higher sensitivity compared with conventional enzyme-linked immunosorbent assay (ELISA), indicating that a simple, rapid, and sensitive immunoassay system could be developed by using KD instead of antibody or antibody fragments.     

HCMV截短UL83基因真核表达重组体的构建、转染及其免疫效力研究

为了构建人巨细胞病毒(HCMV)截短UL83基因真核表达重组体,实现其在Hep-2细胞中的稳定表达,研究该截短UL83基因真核表达重组体免疫效力,采用基因重组的方法,将HCMV AD169株截短UL83基因定向克隆到带有绿色荧光蛋白(GFP)作为报告基因的真核表达载体pEGFP-C1上,构建真核重组表达质粒pEGFP-C1-UL83;脂质体转染至Hep-2细胞中,G418筛选获得稳定表达pp65细胞表达系。经基因测序显示,重组体中截短UL83基因完全正确,RT-PCR和Western blot检测证实其可在Hep-2细胞中稳定表达。用该重组体和其表达产物在HCMV先天性感染小鼠模型上进行免疫保护试验显示,母鼠血清可检测到特异性抗HCMV pp65抗体,效价为:1∶2.51~1∶50.79;子鼠脑组织内未分离出病毒,亦未检测出病毒pp65蛋白抗原表达。初步结果表明,pEGFP-C1-UL83具有较好的免疫原性,可作为DNA疫苗刺激机体产生有效抗体,并具有阻止病毒垂直传播的保护性作用。     

绿色荧光蛋白的原核表达、纯化以及抗体制备

用PCR扩增出绿色荧光蛋白(GFP)基因,插入到pGEX-KG表达载体中,并将构建出的重组质粒命名为pKG- GFP。将重组载体导入大肠杆菌DH10β中,经IPTG诱导产生GST-GFP融合蛋白,同时以可溶蛋白和包涵体两种形式存在。 GST-GFP分子量大约为53kDa,与其理论值大小一致,用亲和层析以及凝血酶处理纯化GFP。纯化的产物经证实具有很好的 均一性。以GFP免疫新西兰家兔,制备多克隆抗体,Westernblotting测定抗血清效价。     

Grafting of protein L-binding activity onto recombinant antibody fragments

Recombinant antibody fragments consisting of variable domains can be easily produced in various host cells, but there is no universal system that can be used to purify and detect them in the free form or complexed with their antigen. Protein L (PpL) is a cell wall protein isolated from Peptostreptococcus magnus, which has been reported to interact with the V-KAPPA chain of some, but not all, antibodies. Here we grafted the V-KAPPA framework region 1 (FR1) sequence of a high-affinity PpL-binding antibody onto single-chain antibody fragments (scFvs), which have no reactivity with PpL. This substitution made it possible to purify and detect scFvs using PpL conjugates. It did not hinder scFv folding and expression in recombinant bacteria, and it did not interfere with their antigen-binding function. We also identified residue 12 as being potentially able to alter PpL binding. This study, therefore, suggests a way of engineering a PpL-binding site on any scFv without interfering with its function. This could provide a universally applicable method both for the rapid purification of functional recombinant antibody fragments and for their detection even when complexed with their antigen without requiring fusion to an epitope Flag.     

Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant

Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.     

Degradation of C-terminal tag sequences on domain antibodies purified from E. coli supernatant

Expression of recombinant proteins often takes advantage of peptide tags expressed in fusion to allow easy detection and purification of the expressed proteins. However, as the fusion peptides most often are flexible appendages at the N- or C-terminal, proteolytic cleavage may result in removal of the tag sequence. Here, we evaluated the functionality and stability of 14 different combinations of commonly used tags for purification and detection of recombinant antibody fragments. The tag sequences were inserted in fusion with the c-terminal end of a domain antibody based on the HEL4 scaffold in a phagemid vector. This particular antibody fragment was able to refold on the membrane after blotting, allowing us to detect c-terminal tag breakdown by use of protein A in combination with detection of the tags in the specific constructs. The degradation of the c-terminal tags suggested specific sites to be particularly prone to proteolytic cleavage, leaving some of the tag combinations partially or completely degraded. This specific work illustrates the importance of tag design with regard to recombinant antibody expression in E. coli, but also aids the more general understanding of protein expression.     

The DNA-binding activity of protein disulfide isomerase ERp57 is associated with the a(') domain

ERp57 belongs to the protein disulfide isomerases, a family of homologous proteins mainly localized in the endoplasmic reticulum and characterized by the presence of a thioredoxin-like folding domain. ERp57 is a protein chaperone with thiol-dependent protein disulfide isomerase and additional activities and recently it has been shown to be involved, in cooperation with calnexin or with calreticulin, in the correct folding of glycoproteins. However, we have demonstrated that the same protein is also present in the nucleus, mainly associated with the internal nuclear matrix fraction. In vitro studies have shown that ERp57 has DNA-binding properties which are strongly dependent on its redox state, the oxidized form being the competent one. A comparison study on a recombinant form of ERp57 and several deletion mutants, obtained as fusion proteins and expressed in Escherichia coli, allowed us to identify the C-terminal a(') domain as directly involved in the DNA-binding activity of ERp57.     

In situ biochemical demonstration that P-glycoprotein is a drug efflux pump with broad specificity

While P-glycoprotein (Pgp) is the most studied protein involved in resistance to anti-cancer drugs, its mechanism of action is still under debate. Studies of Pgp have used cell lines selected with chemotherapeutics which may have developed many mechanisms of resistance. To eliminate the confounding effects of drug selection on understanding the action of Pgp, we studied cells transiently transfected with a Pgp-green fluorescent protein (GFP) fusion protein. This method generated a mixed population of unselected cells with a wide range of Pgp-GFP expression levels and allowed simultaneous measurements of Pgp level and drug accumulation in living cells. The results showed that Pgp-GFP expression was inversely related to the accumulation of chemotherapeutic drugs. The reduction in drug concentration was reversed by agents that block multiple drug resistance (MDR) and by the UIC2 anti-Pgp antibody. Quantitative analysis revealed an inverse linear relationship between the fluorescence of Pgp-GFP and MDR dyes. This suggests that Pgp levels alone limit drug accumulation by active efflux; cooperativity between enzyme, substrate, or inhibitor molecules is not required. Additionally, Pgp-GFP expression did not change cellular pH. Our study demonstrates the value of using GFP fusion proteins for quantitative biochemistry in living cells.     

Expression of green fluorescent protein in Lactococcus lactis

The gfp gene from Aequorea victoria, encoding the green fluorescent protein (GFP) has been expressed in Lactococcus lactis subsp. lactis biovar cremoris MG1363, upon construction and introduction of plasmid pLS1GFP into this host. GFP was monitored in living cells during growth to evaluate its use in molecular and physiological studies. Quantification of the levels of GFP expressed by cultures was feasible by fluorescence spectroscopy. Phase-contrast and fluorescence microscopy allowed us to distinguish, in mixed cultures, lactococcal cells expressing GFP. Our results indicate that GFP can be used as a reporter in L. lactis.