Ca(2+)-triggered coelenterazine-binding protein from Renilla as an enzyme-dependent label for binding assay.

The recombinant Ca(2+)-triggered coelenterazine-binding protein (CBP) from Renilla muelleri was investigated as a biospecifically labeled molecule for in vitro assay applications. The protein was shown to be stable in solutions in the frozen state, as well as stable under heating and to chemical modifications. Conjugates with biotin, oligonucleotide, and proteins were obtained and applied as biospecific molecules in a solid-phase microassay. CBP detection was performed with intact (no modifications were made) Renilla luciferase in the presence of calcium, and the detection limit was found to be 75 amol. Model experiments indicate that this approach shows much promise, especially with regard to the development of multianalytical systems.

The split luciferase complementation assay.

A split luciferase complementation assay to study protein-protein interactions within Arabidopsis protoplasts in 96-well plates is described in this protocol. Two proteins of interest, a bait and prey, which are genetically fused to amino- and carboxy-terminal fragments of Renilla luciferase, are transiently expressed in protoplasts. Physical interactions of these bait and prey proteins reconstitute some of the luciferase activity and result in light emission in the presence of the luciferase substrate. This luminescence is then measured by a microplate luminometer. Amounts of the bait protein accumulated in the protoplasts can be estimated by Western blotting using an antibody that recognizes the amino-terminal fragment of Renilla luciferase. The most advantageous aspect of this assay is its capacity of detecting both association and dissociation of a protein pair of interest in a large-scale format.

Structure based mechanism of the Ca(2+)-induced release of coelenterazine from the Renilla binding protein.

The crystal structure of the Ca(2+)-loaded coelenterazine-binding protein from Renilla muelleri in its apo-state has been determined at resolution 1.8 A. Although calcium binding hardly affects the compact scaffold and overall fold of the structure before calcium addition, there are easily discerned shifts in the residues that were interacting with the coelenterazine and a repositioning of helices, to expose a cavity to the external solvent. Altogether these changes offer a straightforward explanation for how following the addition of Ca(2+), the coelenterazine could escape and become available for bioluminescence on Renilla luciferase. A docking computation supports the possibility of a luciferase-binding protein complex.

Three heterologous proteins simultaneously expressed from a chimeric potyvirus: infectivity, stability and the correlation of genome and virion lengths.

Three heterologous proteins were simultaneously expressed from a chimeric potyvirus Potato virus A (PVA) in Nicotiana benthamiana. The genes for green fluorescent protein of Aequoria victoriae ("G"; 714 nucleotides, nt), luciferase of Renilla reniformis ("L", 933 nt) and beta-glucuronidase of Escherichia coli ("U", 1806 nt) were inserted into the engineered cloning sites at the N-terminus of the P1 domain, the junction of P1 and helper component protein (HC-Pro), and the junction of the viral replicase (NIb) and coat protein (CP), respectively, in an infectious PVA cDNA. The proteins were expressed as part of the viral polyprotein and subsequently released by cleavage at the flanking proteolytic cleavage sites by P1 (one site) or the NIa-Pro proteinase (other sites). The engineered viral genome (pGLU, 13311 nt) was 39.2% larger than wild-type PVA (9565 nt) and infected plants of N. benthamiana systemically. pGLU was stable and expressed all three heterologous proteins, also following the second infection cycle initiated by sap-inoculation of new plants with the progeny viruses. The gene for GUS showed some inherent instabilities, as also reported in other studies. Accumulation of pGLU in infected leaves was lower by a magnitude as compared to the vector viruses pG0U and p0LU used to express two heterologous proteins. Hence, pGLU may have reached the maximum genome size that can still function and complete the PVA infection cycle. Examination of virions by electron microscopy indicated that the virion lengths of PVA chimera with various numbers of inserts were directly proportional to their genome lengths.

Immunohistochemical localization of a retinoic acid-like receptor in nerve cells of two colonial anthozoans (Cnidaria).

Retinoic acid is known to induce vertebrate stem cells to differentiate into a variety of cell types, including neurons. Although retinoic acid was reported to affect morphogenetic pattern specification in the hydrozoan Hydractinia (Müller, W.A., 1984. Retinoids and pattern formation in a hydroid. J. Embryol. Exp. Morph. 81, 253-271) and a retinoid RXR receptor was cloned in the jellyfish Tripedalia (Kostrouch, Z., Kostrouchova, M., Love, W., Jannini, E., Piatigorsky, J., Rall, J.E., 1998. Retinoic acid X receptor in the diploblast, Tripedalia cystophora. Proc. Natl. Acad. Sci. U.S.A. 95, 13442-13447), the cellular targets of retinoids were not investigated. We used Western immunoblotting and immunohistochemistry to investigate the presence and cellular distribution of a RXR-like receptor in the sea pansy Renilla koellikeri and in the staghorn coral Acropora millepora (Cnidaria, Anthozoa). Western blots revealed a 64 kDa protein from a sea pansy extract in a band that co-migrated with a RXR protein from the rat brain. Using antibodies raised against an epitope of human alpha RXR, we visualized putative ectodermal sensory cells in the polyp column of the adult sea pansy. Immunoreactivity was absent in staghorn coral larvae but present in the polyp column of adult colonies in the form of clusters of neuron-like cells in the basiectoderm near the ectoderm-mesoglea interface. These observations suggest that a RXR-like receptor is involved in epithelial nerve cell specification in adult anthozoans and that this role is conserved throughout evolution.

Expression, purification and characterization of calcium-triggered luciferin-binding protein of Renilla reniformis.

The Ca2+-triggered luciferin-binding protein of Renilla reniformis (RLBP) is a non-covalent complex of apoprotein (apoRLBP) and coelenterazine (luciferin). The gene encoding apoRLBP with 552 nucleotides has been synthesized by assembly PCR methods with synthetic oligonucleotides, and the histidine-tagged apoRLBP expressed as a soluble form in the periplasmic space of Escherichia coli cells. The apoRLBP was purified by nickel chelate chromatography and the procedure yielded 18.2mg of recombinant apoRLBP from 80 ml of cultured cells with purity greater than 95%. The purified apoRLBP was converted to RLBP by incubation with coelenterazine in the presence of dithiothreitol and the purity of recombinant RLBP was estimated to be over 95% by comparison with the absorption spectral data of native RLBP. When RLBP mixed with Ca2+, coelenterazine was dissociated from RLBP and was utilized for the luminescence reaction of Renilla luciferase. Also semi-synthetic RLBPs with h-, e-, and Bis-coelenterazines were prepared and characterized.

The 5' leader of the mRNA encoding the mouse neurotrophin receptor TrkB contains two internal ribosomal entry sites that are differentially regulated.

A single internal ribosomal entry site (IRES) in conjunction with IRES transactivating factors (ITAFs) is sufficient to recruit the translational machinery to a eukaryotic mRNA independent of the cap structure. However, we demonstrate that the mouse TrkB mRNA contains two independent IRESes. The mouse TrkB mRNA consists of one of two 5' leaders (1428 nt and 448 nt), both of which include the common 3' exon (Ex2, 344 nt). Dicistronic RNA transfections and in vitro translation of monocistronic RNA demonstrated that both full-length 5' leaders, as well as Ex2, exhibit IRES activity indicating the IRES is located within Ex2. Additional analysis of the upstream sequences demonstrated that the first 260 nt of exon 1 (Ex1a) also contains an IRES. Dicistronic RNA transfections into SH-SY5Y cells showed the Ex1a IRES is constitutively active. However, the Ex2 IRES is only active in response to retinoic acid induced neural differentiation, a state which correlates with the synthesis of the ITAF polypyrimidine tract binding protein (PTB1). Correspondingly, addition or knock-down of PTB1 altered Ex2, but not Ex1a IRES activity in vitro and ex vivo, respectively. These results demonstrate that the two functionally independent IRESes within the mouse TrkB 5' leader are differentially regulated, in part by PTB1.