Recombinant DNA Approach Research Articles

Polyclonal antibodies against the recombinantly expressed coat protein of the Citrus psorosis virus

Psorosis is a damaging disease of citrus that is widespread in many parts of the world. Citrus psorosis virus (CPsV), the type species of the genus Ophiovirus, is the putative causal agent of psorosis. Detection of CPsV by laboratory methods, serology in particular is a primary requirement for large-scale surveys but their production has been impaired by the difficulty of obtaining sufficient clean antigen for immunization. Specific PAbs against coat protein were produced in E. coli using recombinant DNA approach. The full length CP gene fragment was amplified by RT-PCR using total RNA extracted from CPsV infected citrus leaves and CP specific primers. The obtained product (1320bp) was cloned, sequenced and sub-cloned into pET-30(+) expression vector. Expression was induced and screened in different bacterial clones by the presence of the expressed protein (48kDa) and optimized in one clone. Expressed CP was purified using batch chromatography under denaturing conditions. Specificity of expressed protein was demonstrated by ELISA before used as antigen for raising PAbs in mice. Specificity of the raised PAbs to CPsV was verified by ELISA and western blotting. The raised PAbs were showed highly effectiveness in screening by ELISA comparing with the commercial antibodies purchased from Agritest, Valanzano, Italy.The expression of CPsV CP gene in E. coli, production of PAbs using recombinant protein as an antigen, the suitability of these antibodies for use in immunodiagnostics against the CPsV Egyptian isolate have been accomplished in this work.

Polyclonal Antibodies to the Bacterially Expressed Coat Protein of Faba Bean Necrotic Yellows Virus

Specific rabbit polyclonal antibodies against bacterially expressed coat protein of Faba bean necrotic yellows virus (FBNYV, genus Nanovirus) were produced using a recombinant DNA approach. The FBNYV capsid protein (CP) gene located on component 5 was cloned in an expression vector pQE‐9 (Qiagen, QIAGEN Inc., Chatswortch, CA91311, USA). Expression of the CP with an N‐terminal hexahistidine tag in Escheri‐ chia coli M15 cells was induced by adding isopropyl‐3‐D‐1‐thiogalactoside (IPTG) to a final concentration of 2 mM. About 8 mg of bacterially expressed CP (BE‐CP) was purified from 1 litre of bacterial liquid culture using a Ni‐NTA resin column (Qiagen). The expressed CP which migrated as a protein of approximately 23 kDa in sodium dodecyl sulphate (SDS)‐polyacrylamide gel electrophoresis (PAGE) was identified by its strong reaction with polyclonal antibodies produced against FBNYV particles and 2‐5H9 FBNYV‐monoclonal in Western blots. Expressed and purified CP (SDS‐PAGE 23 kDa band) was injected into a white rabbit, using seven intramuscular injections at weekly intervals. The antiserum produced was evaluated for FBNYV detection in double antibody sandwich (DAS)‐enzyme‐linked immunosorbent assay (ELISA), triple antibody sandwich (TAS)‐ELISA, tissue blot immunoassay (TBIA), dot blot, Western blot and goat antimouse coating (GAMC)‐ELISA using 13 different FBNYV monoclonal antibodies. The antiserum raised against the BE‐CP gave strong FBNYV‐specific TBIA reactions and very weak background reactions with non‐infected tissue, similar to those produced by monoclonal antibodies. Furthermore, BE‐CP polyclonal antibody reacted weakly with FBNYV‐infected tissue and strongly with BE‐CP in DAS‐ELISA, but not with FBNYV‐infected tissue in TAS‐ELISA when 13 detecting monoclonal antibodies were used. In addition, BE‐CP polyclonal antibody reacted strongly with BE‐CP in TAS‐ELISA only when 2‐5H9 detecting monoclonal was used. When monoclonals were used as primary antibody and BE‐CP polyclonal as detecting antibody (GAMC‐ELISA), FBNYV‐infected tissue gave moderate reactions with 2‐5H9 and strong reactions with 3‐2E9 monoclonal, whereas BE‐CP gave equally strong reactions with both monoclonals. These results showed that the BE‐CP polyclonal antibody is useful for the detection of FBNYV in infected tissue by TBIA and dot blot tests.

A prodrug approach for delivery of t-PA: construction of the cationic t-PA prodrug by a recombinant method and preliminary in vitro evaluation of the construct.

Previously, we reported a novel prodrug approach, that could lead to targeted thrombolysis without the risk of bleeding. The approach consists of a protein conjugate made of two components: a fibrin targeting antibody (Ab) linked to an anionic heparin, and a plasminogen activator (PA) derivatized with cationic species. These two components are linked by means of an electrostatic interaction. Because the cationic species are small, the modified PA would retain its thrombolytic activity. However, this activity would be inhibited after binding to the counterpart due to the blockage of the PA active site by the appended macromolecules. Because protamine is a clinical antagonist to heparin, it can be used in humans to dissociate the modified PA from its counterpart. Thus, the approach would permit the administration of a fibrin targeting but inactive thrombolytic drug (thereby alleviating the bleeding risk by avoiding systemic generation of plasmin), and subsequently a triggered release of the active drug to the fibrin deposit. In our previous work, we demonstrated the feasibility of the approach by producing a positively charged PA by means of chemical conjugation of a cationic CRRRRRRR peptide with urokinase. In this study, we further extended our work and produced a similar cationic t-PA by means of a recombinant DNA approach; i.e., by fusion of a poly(Arg)7 peptide to the kringle-1 domain of t-PA. Results obtained from the restriction enzyme analysis and the Western blot yielded full identification of this recombinant protein. This recombinant poly(Arg)7-modified-t-PA protein conjugate (termed "rmt-PA" hereafter) completely retained the fibrinolytic activity of the original recombinant, unmodified t-PA (termed "rt-PA" hereafter), as measured by the chromogenic assay and fibrin agar lysis assay. The prodrug and triggered release features of the proposed approach were confirmed by partial inhibition of the plasminogen activating activity of this protein by heparin, and the partial reversal of such inhibition by protamine.

Specific polyclonal antibodies for the obligate plant parasite Polymyxa— a targeted recombinant DNA approach

Highly specific rabbit polyclonal antibodies for the obligate sugar‐beet root parasite, Polymyxa betae, were produced using a novel recombinant DNA approach. Parasite cDNA was selectively isolated from infected roots, expressed in vitro, and the purified protein used to raise antibodies. This produced clean, precisely targeted antibodies, and allowed for rigorous screening of candidate genes and their products at the molecular level prior to animal immunization. This approach selects for genes whose products are highly expressed by the parasite in planta, and five such candidate genes from Polymyxa betae were identified and cloned. Polyclonal antiserum developed using the product of one such gene was found to react specifically with P. betae in sugar‐beet roots and with the closely related Polymyxa graminis in barley roots, and to cross‐react with Plasmodiophora brassicae in cabbage roots, without the need for further purification. No cross‐reaction was detected with protein extracts from potato roots infected by the plasmodiophoromycete Spongospora subterranea. In all cases, there was no interaction with proteins from host plants, or from other microorganisms found in association with uninoculated sugar‐beet, barley, cabbage and potato roots.

Molecular dissection of ligand binding sites on the low density lipoprotein receptor-related protein.

The low density lipoprotein receptor-related protein (LRP) is a large multifunctional receptor that is involved in the cellular uptake of a number of functionally diverse ligands including apoE-rich remnant lipoproteins, lipoprotein lipase, alpha 2-macroglobulin-protease complexes, plasminogen activator-inhibitor complexes, and the active protease tissue-type plasminogen activator. Ligand binding and competition experiments suggest that most LRP ligands bind to specific, independent sites on the large 515-kDa subunit of the receptor. In a previous study (Moestrup, S.K., Holtet, T.L., Etzerodt, M., Thøgersen, H.C., Nykjaer, A., Andreasen, P.A., Rasmussen, H.H., Sottrup-Jensen, L., and Gliemann, J. (1993) J. Biol. Chem. 268, 13691-13696), ligand blotting was used to localize the binding sites for urokinase-type plasminogen activator-plasminogen activator inhibitor-1 (PAI-1) complexes and for alpha 1-macroglobulin to a proteolytic fragment of LRP containing the second cluster of complement-type cysteine-rich repeats. Here, we have used a recombinant DNA approach to express functionally restricted chimeric "LRP-minireceptors" containing two different regions of the extracellular domain of the receptor in cultured cells. Receptor-associated protein, a negative modulator of LRP activity, is bound and internalized by cells transfected with either construct. A minireceptor containing the cluster of eight complement-type cysteine-rich repeats followed by four epidermal growth factor precursor homologous domains binds and internalizes 125I-labeled plasminogen activator-PAI-1 complexes. It also mediates the cellular uptake of the uncomplexed protease tissue-type plasminogen activator (tPA), suggesting that the tPA and PAI-1 binding sites on LRP are in close vicinity and might promote cooperative binding of tPA-PAI-1 complexes. However, alpha 2-macroglobulin is not internalized by this minireceptor suggesting that this ligand requires the presence of a single epidermal growth factor-repeat which is contained in the previously studied proteolytic fragment but is absent from the minireceptor.

Characterization of Recombinant Mouse Perforin Expressed in Insect Cells Using the Baculovirus System

Perforin, a potent pore-forming protein, plays an important role in killer lymphocyte-mediated cytolysis. The studies on perforin, although already extensive, have been hampered by the limited amount of perforin naturally available from killer lymphocytes. In the present study, a full-length mouse perforin was expressed in insect cells using recombinant baculovirus. Recombinant perforin appeared to be functional in terms of lysing erythrocytes and nucleated target cells. These results suggest that the recombinant DNA approach developed in the present study may offer a new way for preparing sufficient amounts of engineered perforin for use in the studies aimed at dissecting the functional domains of the perforin molecule.

At least three distinct B cell epitopes reside in the C‐terminal half of La protein, as determined by a recombinant DNA approach

The La antigen is a nuclear protein that is one of the major target antigens of autoantibodies found in the sera of patients with primary Sjögren's syndrome. The formation of such autoantibodies is therefore likely to reflect the basic immunopathogenesis of this disorder. A recombinant DNA strategy has been used to examine the La protein for sequences that encode autoimmunizing B cell epitopes. We have isolated and characterized a 1.2-kb-long cDNA from a human liver cDNA library encoding a region of the La protein; this region contains 296 amino acids, including the C terminus. A sub-library of recombinant DNA in the expression vector pEX was made from portions of the La cDNA. Individual fusion proteins were tested by immunoblotting and enzyme-linked immunosorbent assay for their ability to react with anti-La autoantibodies contained in sera from patients with Sjögren's syndrome. In this way, we have identified at least three distinct epitopes in the C-terminal half of the La protein. Every anti-La serum tested contained antibodies against all three of the antigenic regions identified. Furthermore, most of the sera display similar ratios between the titers of antibodies with the three kinds of specificity. Our data suggest that the production of anti-La autoantibodies may be antigen driven.

Cloning and expression of the Campylobacter jejuni glyA gene in Escherichia coli

Genetic studies of Campylobacter jejuni are greatly hampered by the lack of genetic markers and an established classical gene transfer mechanism between strains of this species. To facilitate future genetic studies and to provide a recombinant DNA approach for analyzing genes of C. jejuni, we constructed an extensive genomic library of a pathogenic C. jejuni strain TGH9011 (serotype 0:3) using pBR322. We report the isolation of a number of recombinant plasmids containing the complete structural gene of glyA, that encodes serine hydroxymethyltransferase (SHMT) of C. jejuni. Escherichia coli cells containing this multicopy recombinant plasmid with the glyA gene produce high levels of SHMT. The SHMT-encoding fragment was identified by subcloning and functional complementation. The expression of the C. jejuni glyA gene was probably via transcription initiated from its own promoter.

Topological mapping of complement component C9 by recombinant DNA techniques suggests a novel mechanism for its insertion into target membranes.

cDNA molecules coding for mouse and trout C9 have been isolated and the derived amino acid sequences compared with that of human C9. Regions of high homology between the closely related species (mouse and human) correlate with putative domains in the protein structure supporting a model of C9 having five globular domains. Comparison between the more distant species (trout and human) suggests regions of particular importance to C9 structure and function. In addition the three related sequences allow the secondary structure to be predicted with more confidence and we have tested the prediction by mapping surface features of the protein. Reported here is a recombinant DNA approach to fine mapping of antibody epitopes. Two of the putative domains of C9 are connected by a stretch of about 40 amino acid residues in which features characteristic of individual conformational forms of C9 are concentrated. We suggest that this region might act as a hinge allowing the rearrangement of globular domains necessary for membrane insertion. In the membrane inserting domain one highly conserved sequence has the potential to form an amphipathic alpha-helix once it is buried in the lipid bilayer. These features suggest a novel mechanism for the irreversible, post-translational insertion of C9 into target membranes.

Recombinant DNA approach for defining the primary structure of monoclonal antibody epitopes: The analysis of a conformation-specific antibody to myosin light chain 2

A monoclonal antibody (MF5), capable of recognizing a divalent cation-induced conformational change in myosin light chain 2 (LC2f), has been used to screen a cDNA library constructed in the expression vector λgt11. A clone has been isolated that contains the whole coding sequence of this myosin subunit. The light chain was synthesized as a fusion peptide linked to β-galactosidase by ten amino acids encoded in the 5′ untranslated region of its mRNA. Seven imperfect repeats were identified in the 3′ untranslated region of the mRNA. The amino acids conferring specificity on the MF 5 epitope were established by first determining the nucleotide sequence of shorter subclones that expressed the epitope and then eliminating those amino acid residues shared by cardiac myosin LC2, which was unreactive with this antibody. The epitope, which becomes accessible to MF 5 upon removal of bound divalent cations, resides at the junction between the first λ-helical domain and the metal binding site. Theoretically, this approach can be used to define the primary structure of most protein epitopes.

The role of a signaling protein in bacterial sensing: behavioral effects of increased gene expression.

A recombinant DNA approach has been used to study intracellular signaling in the bacterial sensing system. The Escherichia coli cheY gene, whose function is unknown, has been subcloned behind the synthetic inducible tac promoter. The resulting plasmid directs the synthesis of the Y protein in response to isopropyl beta-D-thiogalactoside, independent of its usual operon control. When this construct was introduced into wild-type and mutant cells, the Y protein caused a clockwise rotational bias in the flagellar motors. This effect was observed even in heavily biased counterclockwise strains lacking most of the central chemotaxis processing genes. The results show that the Y protein has a direct influence on flagellar rotation not requiring other processing genes of the sensing system. The Y protein appears to bind directly to a part of the flagellar motor, probably the flaA gene product, and it is probably the key element in biasing the motor toward the clockwise rotational direction.

A model for the structure of fumarate reductase in the cytoplasmic membrane of Escherichia coli.

By a recombinant DNA approach we have prepared Escherichia coli cytoplasmic membranes that are highly enriched in the terminal electron transfer enzyme fumarate reductase. This enzyme is composed of four nonidentical subunits in equal molar ratio. A 69,000-dalton covalent flavin-containing subunit and a 27,000-dalton nonheme iron-containing subunit make up a membrane extrinsic catalytic domain. Two very hydrophobic subunits of 15,000 and 13,000 daltons make up the hydrophobic membrane anchor domain. Electron microscopy of negatively stained membranes shows a characteristic knob-and-stalk-type structure composed of the catalytic domain. The anchor polypeptides have been analyzed for hydrophobic segments and alpha-helical content and a model for their organization within the lipid bilayer is presented. The results reviewed in this paper suggest a model for the fumarate reductase complex in the cytoplasmic membrane.

Recombinant DNA approach to neurogenetic disorders

The development of recombinant DNA techniques over the past 5 years has revolutionized many fields in biology. The application of these techniques in the neurosciences has so far involved the isolation and characterization of sequences coding for neuroactive polypeptides via the isolation of a DNA copy of the mRNA coding for the polypeptide in question. However, there are numerous examples of alleles of single loci which affect the organization or performance of the nervous system of the organism, including some of the major human neurogenetic disorders, for which the relevant polypeptide choice is unknown. Recombinant DNA techniques can also provide an alternative approach, via the application of genetic linkage methods, for identifying the DNA sequences responsible for the abnormal phenotype in these cases. In this article we focus on the analysis of human neurogenetic disorders such as Huntington's disease although the general principles may also be applicable to other organisms in which genes affecting neurological phenotypes have been identified.