Antibody-induced oligomerization and activation of an engineered reporter enzyme.
ABSTRACT: Our objective is to produce a protein biosensor (or molecular switch) that is specifically activated in solution by a monoclonal antibody. Many effector-dependent enzymes have evolved in nature, but the introduction of a novel regulatory mechanism into a normally unregulated enzyme poses a difficult design problem. We used site-saturation mutagenesis and screening to generate effector-activated variants of the reporter enzyme beta-glucuronidase (GUS). The specific activity of the purified epitope-tagged GUS variant was increased by up to approximately 500-fold by the addition of an equimolar concentration of a monoclonal antibody. This molecular switch is modular in design, so it can easily be re-engineered for the detection of other peptide-specific antibodies. Such antibody-activated reporters could someday enable point-of-care serological assays for the rapid detection of infectious diseases.
Project description:A ?-glucuronidase variant, GUS-TR3337, that was obtained by directed evolution exhibited higher thermostability than the wild-type enzyme, GUS-WT. In this study, the utility of GUS-TR337 as an improved reporter was evaluated. The corresponding gus-tr3337 and gus-wt genes were independently cloned in a plant expression vector and introduced into Arabidopsis thaliana. With 4-MUG as a substrate, plants containing the gus-wt gene showed no detectable ?-glucuronidase activity after exposure to 60°C for 10 min, while those hosting the gus-tr3337 gene retained 70% or 50% activity after exposure to 80°C for 10 min or 30 min, respectively. Similarly, in vivo ?-glucuronidase activity could be demonstrated by using X-GLUC as a substrate in transgenic Arabidopsis plants hosting the gus-tr3337 gene that were exposed to 80°C for up to 30 min. Thus, the thermostability of GUS-TR3337 can be exploited to distinguish between endogenous and transgenic ?-glucuronidase activity, which is a welcome improvement in its use as a reporter.
Project description:To avoid the unregulated overexpression of the exogenous genes, specific or inducible expression is necessary for some exogenous genes in transgenic plants. But little is known about organ- or tissue-specific promoters in maize. In the present study, the expression of a maize pentatricopeptide repeat (PPR) protein encoding gene, GRMZM2G129783, was analyzed by RNA-sequencing data and confirmed by quantitative real time PCR. The results showed that the PPR GRMZM2G129783 gene specifically expressed in vegetative organs. Consequently, a 1830 bp sequence upstream of the start codon of the promoter for GRMZM2G129783 gene was isolated from maize genome (P 1830 ). To validate whether the promoter possesses the vegetative organ-specificity, the full-length and three 5'-end deletion fragments of P 1830 of different length (1387, 437, and 146 bp) were fused with glucuronidase (GUS) gene to generate promoter::GUS constructs and transformed into tobacco. The transient expression and fluorometric GUS assay in transgenic tobacco showed that all promoter could drive the expression of the GUS gene, the -?437 to -?146 bp region possessed some crucial elements for root-specific expression, and the shortest and optimal sequence to maintain transcription activity was 146 and 437 bp in length, respectively. These results indicate that the promoter of the PPR GRMZM2G129783 gene is a vegetative organ-specific promoter and will be useful in transgenic modification of commercial crops for moderate specific expression after further evaluation in monocotyledons.
Project description:It is known that digital counting of fluorescent signals generated in many small compartments can significantly improve the detection sensitivity of the enzyme-linked immunosorbent assay (ELISA). However, the reported digital ELISA systems need extensive washing steps to remove background signal, which hampers their performance. To tackle this problem, we developed a vesicle (Protocell) array wherein binding of an external protein analyte is coupled to signal amplification and intra-vesicular fluorescence readout. We chose ?-glucuronidase (GUS) as a reporter enzyme as its function requires assembly of four subunits through dimerization of a pair of dimers that can be inhibited by a set of interface mutations. Using a thermostabilized GUS mutant IV-5, we screened out an interface mutant (M516K, F517W) to create IV5m - a mutant with high thermostability and activity conditional on induced dimerization. After tethering a short N-terminal tag and transmembrane (TM) sequences, the fusion protein was expressed by cell-free protein synthesis inside protocells. When a corresponding tag-specific antibody was applied outside of the protocells, a clear increase in GUS activity was observed inside vesicles by adding fluorescent substrate, probably due to spontaneous integration of the tagged TM protein into the vesicles and dimerization by the antibody bound to the displayed tag. Furthermore, using flow cytometry, quantitative digital read out was obtained by counting fluorescent protocells exposed to varying concentrations of external antibodies that included Trastuzumab. Additionally, through use of an anti-caffeine VHH-SpyCatcher fusion protein, caffeine could be detected using SpyTag-fused TM-IV5m protein expressed in protocells, suggesting utility of this platform for detection of diverse antigen types.
Project description:The gene encoding the rice transcription factor, REB (rice endosperm bZIP) was cloned from a bacterial artificial chromosome library of rice. The cloned 6,227-bp-long Reb gene is composed of six exons and five introns and is flanked by a 1.2-kb 5' promoter and a 1.2-kb 3' terminator region. The function of the Reb gene was explored by a transient assay by using a rice immature endosperm system. The effector constructs containing the native gene or fusion genes linking Reb to the rice actin (Act) or globulin (Glb) gene promoters and the reporter gene construct Glb-beta-glucuronidase (GUS) were used in this study. When these effector constructs were cotransferred with the reporter uidA gene encoding GUS under the control of the Glb promoter into immature rice endosperm cells, the Glb promoter was activated. The transient GUS expression was 2.0 to 2.5-fold higher with the effector construct than without. When the upstream activation sequence containing the GCCACGT(A/C)AG motifs of the Glb promoter was deleted, the activation by REB was abolished. On the other hand, a gain-of-function experiment showed that inserting the upstream activation sequence into the glutelin-1 (Gt1) promoter made it responsive to activation by REB. When cotransformed with Reb gene, mature transgenic rice grains containing the human lysozyme gene driven by the Glb promoter produced 3.7-fold more lysozyme. Accumulation of recombinant lysozyme in mature seed ranged from 30.57 to 279.61 microg.mg(-1) total soluble protein in individual transformants from 30 independent transformation events. Thus, our results show that REB is not only a transcriptional activator, it can also be used to increase the expression of recombinant protein in transgenic rice grains.
Project description:Glycosyl hydrolases hydrolyze the glycosidic bond in carbohydrates or between a carbohydrate and a non-carbohydrate moiety. beta-glucuronidase (GUS) is classified under two glycosyl hydrolase families (2 and 79) and the family-2 beta-glucuronidase is reported in a wide range of organisms, but not in plants. The family-79 endo-beta-glucuronidase (heparanase) is reported in microorganisms, vertebrates and plants. The E. coli family-2 beta-glucuronidase (uidA) had been successfully devised as a reporter gene in plant transformation on the basis that plants do not have homologous GUS activity. On the contrary, histochemical staining with X-Gluc was reported in wild type (non-transgenic) plants. Data shows that, family-2 beta-glucuronidase homologous sequence is not found in plants. Further, beta-glucuronidases of family-2 and 79 lack appreciable sequence similarity. However, the catalytic site residues, glutamic acid and tyrosine of the family-2 beta-glucuronidase are found to be conserved in family-79 beta-glucuronidase of plants. This led to propose that the GUS staining reported in wild type plants is largely because of the broad substrate specificity of family-79 beta-glucuronidase on X-Gluc and not due to the family-2 beta-glucuronidase, as the latter has been found to be missing in plants.
Project description:Arabidopsis encodes six nuclear actin-related proteins (ARPs), among them ARP8 is unique in having an F-box domain and an actin homology domain. Analysis of the ARP8 promoter-beta-glucuronidase (GUS) fusion suggests that ARP8 is ubiquitously expressed in all organs and cell types. Immunocytochemical analysis with ARP8-specific monoclonal antibodies revealed that ARP8 protein is localized to the nucleolus in interphase cells and dispersed in the cytoplasm in mitotic cells. The cell cycle-dependent subcellular patterns of distribution of ARP8 are conserved in other members of Brassicaceae. Our findings provide the first insight into the possible contributions of plant ARP8 to nucleolar functions.
Project description:Root-knot nematodes are sedentary biotrophic endoparasites that maintain a complex interaction with their host plants. Nematode effector proteins are synthesized in the oesophageal glands of nematodes and secreted into plant tissue through a needle-like stylet. Effectors characterized to date have been shown to mediate processes essential for nematode pathogenesis. To gain an insight into their site of action and putative function, the subcellular localization of 13 previously isolated Meloidogyne incognita effectors was determined. Translational fusions were created between effectors and EGFP-GUS (enhanced green fluorescent protein-?-glucuronidase) reporter genes, which were transiently expressed in tobacco leaf cells. The majority of effectors localized to the cytoplasm, with one effector, 7H08, imported into the nuclei of plant cells. Deletion analysis revealed that the nuclear localization of 7H08 was mediated by two novel independent nuclear localization domains. As a result of the nuclear localization of the effector, 7H08 was tested for the ability to activate gene transcription. 7H08 was found to activate the expression of reporter genes in both yeast and plant systems. This is the first report of a plant-parasitic nematode effector with transcriptional activation activity.
Project description:A method has been established to sequentially delete combinations of genes from the ASFV genome to test the effect on virus replication and host responses to infection. Initially the ASFV genes MGF505 2R and MGF505 3R and a truncated MGF360 9L gene were deleted from the genome of the tissue-culture adapted ASFV strain BA71V and replaced with bacteriophage loxP sequences flanking the beta-glucuronidase (GUS) marker gene to create recombinant virus V?MGF-GUS. Subsequently the GUS gene was removed by site-specific recombination between the two loxP sites involving expression of the bacteriophage Cre recombinase enzyme to create recombinant virus V?MGF?GUS. The EP402R and EP153R genes were subsequently deleted from the genome of V?MGF?GUS, using the same GUS marker gene, to construct virus V?MGF?CD2-Lectin-GUS. These sequential deletions of ASFV genes were shown not to alter virus replication significantly.
Project description:Crohn's disease, an incurable chronic inflammatory bowel disease, has been attributed to both genetic predisposition and environmental factors. A dysbiosis of the gut microbiota, observed in numerous patients but also in at least one hundred unaffected first-degree relatives, was proposed to have a causal role. Gut microbiota ?-D-glucuronidases (EC 220.127.116.11) hydrolyse ?-D-glucuronate from glucuronidated compounds. They include a GUS group, that is homologous to the Escherichia coli GusA, and a BG group, that is homologous to metagenomically identified H11G11 BG and has unidentified natural substrates. H11G11 BG is part of the functional core of the human gut microbiota whereas GusA, known to regenerate various toxic products, is variably found in human subjects. We investigated potential risk markers for Crohn's disease using DNA-sequence-based exploration of the ?-D-glucuronidase loci (GUS or Firmicute H11G11-BG and the respective co-encoded glucuronide transporters). Crohn's disease-related microbiomes revealed a higher frequency of a C7D2 glucuronide transporter (12/13) compared to unrelated healthy subjects (8/32). This transporter was in synteny with the potential harmful GUS ?-D-glucuronidase as only observed in a Eubacterium eligens plasmid. A conserved NH2-terminal sequence in the transporter (FGDFGND motif) was found in 83% of the disease-related subjects and only in 12% of controls. We propose a microbiota-pathology hypothesis in which the presence of this unique ?-glucuronidase locus may contribute to an increase risk for Crohn's disease.
Project description:We had previously reported that the InMYB1 promoter, the 1023 bp upstream region of InMYB1, works petal-specifically in various dicot plants by recognizing petal identity at a cellular level. To determine the petal-specific region in the InMYB1 promoter, Arabidopsis plants harboring InMYB1_1023b::GUS (?-glucuronidase), InMYB1_713b::GUS, InMYB1_506b::GUS, InMYB1_403b::GUS, InMYB1_332b::GUS, InMYB1_200b::GUS and InMYB1_140b::GUS were produced and confirmed a shortest region, which has the petal-specific promoter activity by using histochemical GUS assay. Petal-specific GUS staining was not observed in the Arabidopsis plants transformed with InMYB1_200b::GUS and InMYB1_140b::GUS, but observed in transgenic Arabidopsis plants harboring from InMYB1_1023b::GUS to InMYB1_332b::GUS. cDNA sequence of InMYB1 shows that 120 bp upstream region of InMYB1 is 5' untranslated region, suggesting that the 332-121 bp upstream region of InMYB1 contains an important element for petal-specific gene expression. In the Arabidopsis harboring the InMYB1_332-121b×3_TATA_?::GUS, petal-specific GUS staining was observed and the staining was stronger than in the Arabidopsis harboring InMYB1_1023b::GUS. This result shows that the 332-121 bp region is enough and essential for the petal specificity and the InMYB1_332-121b×3_TATA_? could be used for the molecular breeding of floricultural crops.