Isolation and characterization of a polyol-responsive monoclonal antibody useful for gentle purification of Escherichia coli RNA polymerase.

A modified enzyme-linked immunosorbent assay (ELISA) was used to screen monoclonal antibodies (MAbs) that react with Escherichia coli RNA polymerase for the ability to release the RNA polymerase in the presence of a low molecular weight polyhydroxylated compound (polyol) and a non-chaotropic salt. This assay, termed the ELISA-elution assay, identified 19 presumptive "polyol-responsive" MAbs out of a total of 218 antigen-specific MAbs screened. One of these MAbs, designated NT73, was examined in detail for the ability to release the antigen in response to various combinations of polyol and salt. Using NT73 conjugated to Sepharose, highly active RNA polymerase could be prepared rapidly by a single immunoaffinity chromatography step, replacing two lengthy chromatographic steps in our conventional purification procedure. Because NT73 reacts with the beta' subunit of RNA polymerase, a mixture of the core polymerase and holoenzyme was recovered from the immunoaffinity column. The holoenzyme (E sigma 70) could be separated from the core polymerase by subsequent chromatography on a Mono Q column. This demonstrates that polyol-responsive MAbs can be easily identified and characterized by the ELISA-elution assay. The use of polyol-responsive MAbs provides a means of adapting immunoaffinity chromatography to the purification of labile proteins.     

Identification of the epitope for a highly cross-reactive monoclonal antibody on the major sigma factor of bacterial RNA polymerase.

A highly cross-reactive monoclonal antibody (MAb), 2G10, was found to react in a conserved region of Escherichia coli RNA polymerase sigma70. The epitope was localized to amino acids 470 to 486, which included part of conserved region 3.1. The epitope for MAb 3D3, a MAb which maps close to the 2G10 epitope, was also determined.     

Purification of eukaryotic RNA polymerase II by immunoaffinity chromatography. Elution of active enzyme with protein stabilizing agents from a polyol-responsive monoclonal antibody

Active eukaryotic RNA polymerase II (RNAP II) was purified by immunoaffinity chromatography, using a monoclonal antibody (mAb) that reacts with the highly conserved heptapeptide repeat of the largest subunit. This mAb (designated SWG16) was conjugated to CNBr-activated Sepharose and used to purify RNAP II from wheat germ and calf thymus. The subunit composition of the immunoaffinity-purified enzyme was essentially the same as RNAP II purified by conventional chromatography except that it contained only the form with the unproteolyzed largest subunit. Active enzyme could be eluted from the SWG16-Sepharose, at pH 7.9, with combinations of low molecular weight polyols and nonchaotropic salts. The superior eluting procedure used combinations of ethylene glycol (30-40%) and ammonium sulfate (0.5-0.75 M). Active enzyme also could be eluted with a synthetic peptide containing four repeats of the heptapeptide; however, the peptide was not as effective as the polyol and salt combinations for eluting the enzyme. This mAb should be useful for purifying RNAP II from many eukaryotic species. Because the elution of enzyme from the immunoadsorbent seems to be dependent upon the presence of a polyol, this antibody is referred to as a "polyol-responsive mAb." A procedure that helps to identify a polyol-responsive mAb and to optimize the eluting conditions is described. Polyol-responsive mAbs might have broad applicability to the purification of many labile enzymes by immunoaffinity chromatography.     

Expression and purification of a single-chain variable fragment antibody derived from a polyol-responsive monoclonal antibody

A previously described polyol-responsive monoclonal antibody (PR-mAb) was converted to a single-chain variable fragment (scFv). This antibody, PR-mAb NT73, reacts with the beta' subunit of Escherichia coli RNA polymerase and has been used for the immunoaffinity purification of polymerase. mRNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) were used as the template for cDNA synthesis. The sequences were joined by the addition of a "linker" sequence and then cloned into several expression vectors. A variety of expression plasmids and E. coli hosts were used to determine the optimal expression system. Expression was highest with the pET22b(+) vector and the Rosetta(DE3)pLysS host strain, which produced approximately 60 mg purified His-tagged scFv per liter of culture (3.3 g wet weight cells). Although the production of soluble scFv was preferred, overproduced scFv formed inclusion bodies under every expression condition. Therefore, inclusion bodies had to be isolated, washed, solubilized, and refolded. The FoldIt protein refolding kit and enzyme-linked immunosorbent assay were sequentially used to determine the optimal refolding conditions that would produce active His-tagged scFv. Immobilized metal affinity chromatography was used for the final purification of the refolded active scFv. The polyol-responsiveness of the scFv was determined by an ELISA-elution assay. Although the scFv loses considerable affinity for its antigen, it maintains similar polyol-responsiveness as the parent monoclonal antibody, PR-mAb NT73.     

The epitope for the polyol-responsive monoclonal antibody 8RB13 is in the flap-domain of the beta-subunit of bacterial RNA polymerase and can be used as an epitope tag for immunoaffinity chromatography

Polyol-responsive monoclonal antibodies (PR-mAbs) are useful for the purification of proteins in an easy, one step immunoaffinity step. These antibodies allow for gentle purification of proteins and protein complexes using a combination of a low molecular weight polyhydroxylated compound (polyol) and a nonchaotrophic salt in the eluting buffer. mAb 8RB13 has been characterized as one of these PR-mAbs and has been used to purify RNA polymerase from five species of bacteria. Here the epitope for 8RB13 has been identified as PEEKLLRAIFGEKAS, a sequence that is highly conserved in the β-subunit of bacterial RNA polymerase. This sequence is located in the "beta-flap" domain of RNA polymerase (and essentially comprises the "flap-tip helix"), an important binding site for sigma70. This location explains why only the core RNAP is purified using this mAb. This amino acid sequence has been developed into an epitope tag that can be used to purify a target protein from either bacterial or eukaryotic cells when genetically fused to a protein of interest.     

Rapid purification of the oxygenase component of toluene dioxygenase from a polyol-responsive monoclonal antibody.

A monoclonal antibody designated 302 beta that is specific for the beta subunit of the oxygenase component (ISPTOL) of toluene dioxygenase from Pseudomonas putida F1 was used to prepare an immunoaffinity column. ISPTOL in cell extracts of Escherichia coli JM109(pDTG611) bound to the column, and an enzyme-linked immunosorbent elution-screening assay with different combinations of polyols and kosmotropic anions was used to determine the conditions necessary for recovery of active enzyme. Elution from an 8-ml antibody column with 50 mM 2-(N-morpholino)ethanesulfonate buffer (pH 6.8) containing 50% ethylene glycol, 1.0 M ammonium sulfate, 1.0 mM dithiothreitol, and 0.2 mM ferrous ammonium sulfate gave approximately 2 mg of ISPTOL with a specific activity that was more than 300 times the specific activity previously obtained.     

A simple procedure for resolution of Escherichia coli RNA polymerase holoenzyme from core polymerase.

The association constant, K_A, for myosin subfragment-1 binding to actin was measured as a function of ionic strength [KCl, LiCl, and tetramethylammonium chloride (TMAC)]and temperature by the method of time-resolved fluorescence depolarization. The following thermodynamic values were obtained from solutions of 0.20 × 10^?6m S-1, 1.00 × 10^?6m actin in 0.15 m KCl, pH 7.0, at 25 °C: ΔG ° = ?39 ± 1 kJ M^?1, ΔH⁰ = 44 ± 2 kJ M^?1 and $\text{ΔS}{}^0\text{= 0.28 ± 0.01 kJ}\text{M}{}^{\mathrm{?1}}{}^0\text{K}{}^{\mathrm{?1}}$. For measurements in KCl (0.05 to 0.60 m), In $\text{K}{}_{\mathrm{a}}\text{= ?8.36 (KCl)}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$. Thus, the binding is endothermic and strongly inhibited by high ionic strength. When KCl was replaced by LiCl or TMAC the ionic effects on the binding were cation specific. The nature of actin-(S-1) binding in the rigor state is discussed in terms of these results.     

A functionally unique anti-CD3 monoclonal antibody cross-reactive with basal keratinocytes

Monoclonal antibodies (mAb) 147, 446, and 454 each recognize different epitopes of CD3. The CD3 epitope recognized by mAb 446 is functionally unique for the T cell. In contrast to mAb 147 and 454, mAb 446 induces modulation of surface CD3 with delayed kinetics and, hence, is impaired in inducing a refractory state in the T cell to subsequent anti-CD3-induced helper function. MAb 446 (but not other anti-CD3 mAb, including mAb 147, 454, OKT3, and anti-Leu4) recognizes a cytoplasmic determinant within basal keratinocytes. Extraction of keratinocytes with nonionic detergent and 2 M NaCl abolished subsequent staining with mAb 446 but enhanced subsequent staining with anti-keratin mAb, suggesting that this cross-reactive determinant is not keratin. Immunoprecipitation of internally labeled keratinocytes with the anti-CD3 mAb 147 and 446 failed to reveal specific bands, whereas these same mAb immunoprecipitated specific bands from internally labeled CD3+ Jurkat cells corresponding to previously identified CD3 subunits, suggesting that the cross-reactive determinant in keratinocytes is also not CD3. The cross-reactivity is not species specific, in that mAb 446 stained a mouse keratinocyte line, nor is it absolutely keratinocyte specific, in that mAb 446 stained one of the two nonkeratinocyte human epithelial cell lines tested. This study raises the possibility that perturbation of unique CD3 epitopes may have unique effects on T cell surface events and subsequent activation and function.     

In vitro expression and monoclonal antibody of RNA-dependent RNA polymerase for infectious bursal disease virus

VP1, the RNA-dependent RNA polymerase of infectious bursal disease virus (IBDV), has been suggested to play an essential role in the replication and translation of viral RNAs. In this study, we first expressed the complete VP1 protein gene in Escherichia coli (E. coli), and then the produced polyclonal antibody and four monoclonal antibodies (mAbs) to recombinant VP1 protein (rVP1) were shown to bind the IBDV particles in chicken embryo fibroblast and Vero cells. The epitopic analysis showed that mAbs 1D4 and 3C7 recognized respectively two distinct antigenic epitopes on the rVP1 protein, but two pair of mAbs 1A2/2A12 and 1E1/1H3 potentially recognized another two topologically related epitopes. Immunocytochemical stainings showed that VP1 protein formed irregularly shaped particles in the cytoplasm of the IBDV-infected cells. These results demonstrated that the mAbs to rVP1 protein could bind the epitopes of IBDV particles, indicating that the rVP1 protein expressed in E. coli was suitable for producing the mAb to VP1 protein of IBDV, and that the cytoplasm could be the crucial site for viral genome replication of IBDV.     

Production and characterization of a monoclonal antibody cross-reactive with most group A trichothecenes

A monoclonal antibody cross-reactive with most group A trichothecenes was produced by fusion of P3/NS-1/1-AG4-1 myeloma cells with spleen cells isolated from a BALB/c mouse that had been immunized with 3-acetyl-neosolaniol-hemisuccinate conjugated to bovine serum albumin. One stable clone, H159B1D5, which produced monoclonal antibody that bound with both T-2 toxin and diacetoxyscirpenol (DAS) was obtained after subcloning. Enzyme-linked immunosorbent assay (ELISA) revealed that the antibody belongs to the immunoglobulin G1 (kappa chain) isotype and had binding constants of 2.81 x 10(9), 1.05 x 10(9), and 1.57 x 10(8) liters per mole for T-2 tetraol tetraacetate, T-2 toxin, and DAS, respectively. The relative cross-reactivities of the antibody with T-2 tetraol tetraacetate, T-2 toxin, and DAS were 200, 100, and 20, respectively, with tritiated T-2 toxin as the marker ligand. The relative cross-reactivities for the above toxins were 667, 100, and 73, respectively, with tritiated DAS as the marker ligand. No cross-reaction with HT-2 and deoxynivalenol triacetate was observed in either system. By using this monoclonal antibody, an indirect ELISA for analysis of T-2 toxin was also developed. The linear portion of the standard curve for analysis of T-2 toxin in each analysis by radioimmunoassay and ELISA was in the range of 0.1 to 2 ng and 0.05 to 1.0 ng, respectively.     

A soluble biotinyl-alpha-amanitin affinity probe for the isolation of RNA polymerase B

Utilizing the 6'-hydroxyindole moiety of alpha-amanitin for substitution, biotinyl-alpha-amanitin has been synthesized to use as a soluble affinity probe for the isolation of RNA polymerase B from mammalian cell culture. The synthetic biotinyl-alpha-amanitin remains a potent inhibitor of RNA polymerase B having a Ki of 4.1 X 10(-8) M as compared with a Ki of 5 X 10(-9) M for natural alpha-amanitin. RNA polymerase B complexed with biotinyl-alpha-amanitin can be isolated on Bio-Gel P300 polyacrylamide gel beads to which avidin has been attached. RNA polymerase B may then be released from the complex by treatment with sodium dodecyl sulfate or by monochromatic irradiation at 314 nm which destroys the anatoxin moiety. We have used this affinity probe to analyze the subunit composition of RNA polymerase B from various mouse myeloma cell lines. We believe that the biotinyl-alpha-amanitin may be very useful for the isolation of factors which associate with RNA polymerase B; e.g., we have substantiated that actin can be associated with RNA polymerase.     

A high-affinity monoclonal antibody against the FLAG tag useful for G-protein-coupled receptor study

The FLAG sequence (DYKDDDDK) is an artificial sequence widely used to detect, quantify, and purify proteins expressed as FLAG-fusion proteins. Several highly specific monoclonal antibodies for FLAG are commercially available; however, they are not always sensitive enough to detect proteins expressed at low levels and can give rise to unacceptable levels of background signal when used for immunostaining in vitro and in vivo. The current study reports the successful establishment of hybridoma cells that produce an extremely high-affinity antibody to FLAG, namely 2H8 Ab. 2H8 Ab stained FLAG-tagged G-protein-coupled receptors more strongly than commercially available antibodies in both flow cytometry and immunostaining experiments with no background staining. 2H8 was sensitive enough to detect FLAG-tagged G-protein-coupled receptors and soluble proteins in crude preparations, which could not be achieved using commercially available antibodies. Only 10 ng of 2H8 Ab was required to immunoprecipitate FLAG-tagged G-protein-coupled receptors from cell lysates. Of note, 2H8 stained FLAG-tagged BLT2, a low-affinity leukotriene B4 receptor, expressed in vivo in the small intestine of mice under control of the villin promoter. Thus, 2H8 Ab is a promising tool for analyzing various FLAG-fusion proteins, particularly G-protein-coupled receptors, both in vitro and in vivo.     

RNA species that replicate with DNA-dependent RNA polymerase from Escherichia coli

An RNA that replicates with core RNA polymerase from E. coli and the substrates ATP, CTP, ITP, and UTP, was selected from a random poly(A,U,I,C) library and named EcorpI. Another replicating RNA, EcorpG, was obtained by template-free incubation of holo RNA polymerase and the substrates ATP, CTP, GTP, and UTP. Both RNA species showed typical autocatalytic RNA amplification profiles with replication rates in the range of other RNA replicons. The replication products were heterogeneous in length; the different lengths appeared to be different replication intermediates. Both RNA were single-stranded with much internal base-pairing but low melting points. Their sequences were composed by permutations of certain sequence motives in both polarities separated by short oligo(A) and oligo(U) clusters. There was evidence for 3'-terminal elongation on an intramolecular template. No double-stranded RNA was found, even though base-pairing is certainly the underlying basis of the replication process. The reaction was highly sensitive: a few RNA strands were sufficient to trigger an amplification avalanche.