Fusion Protein Technology Research Articles

Peptide affinity chromatography media that bind N pro fusion proteins under chaotropic conditions

To design a generic purification platform and to combine the advantages of fusion protein technology and matrix-assisted refolding, a peptide affinity medium was developed that binds inclusion body-derived N pro fusion proteins under chaotropic conditions. Proteins were expressed in Escherichia coli using an expression system comprising the autoprotease N pro from Pestivirus, or its engineered mutant called EDDIE, with C-terminally linked target proteins. Upon refolding, the autoprotease became active and cleaved off its fusion partner, forming an authentic N-terminus. Peptide ligands binding to the autoprotease at 4 M urea were screened from a combinatorial peptide library. A group of positive peptides were identified and further refined by mutational analysis. The best binders represent a common motif comprising positively charged and aromatic amino acids, which can be distributed in a random disposition. Mutational analysis showed that exchange of a single amino acid within the peptide ligand caused a total loss of binding activity. Functional affinity media comprising hexa- or octapeptides were synthesized using a 15-atom spacer with terminal sulfhydryl function and site-directed immobilization of peptides derivatized with iodoacetic anhydride. The peptide size was further reduced to dipeptides comprising only one positively charged and one aromatic amino acid. Based on this, affinity media were prepared by immobilization of a poly amino acid comprising lysine or arginine, and tryptophan, phenylalanine, or tyrosine, respectively, in certain ratios. Binding capacities were in the range of 7–15 mg protein mL −1 of medium, as could be shown for several EDDIE fusion proteins. An efficient protocol for autoproteolytic cleavage using an on-column refolding method was implemented.

Integrin targeted drug and gene delivery

Importance of the field: Recently, there has been substantial progress in the development of integrin targeted pharmaceuticals and drug delivery systems. Integrin is an important member in the cell adhesion molecule family, which is involved in regulation of complex biological conditions, from keeping normal physiological activities to causing cellular dysfunction in diseased cells. Hence, it is timely to summarize the recent developments in integrin targeted drug and gene delivery systems to understand better their advantages and limitations.Areas covered in this review: In this review, advances in the discovery and clinical trials of these integrin antagonists against different integrin subunits are summarized and discussed. Besides using integrin inhibitor as a single therapeutic agent, integrin antagonists that were conjugated to cytotoxic drugs by synthetic chemistry or coupled to biomacromolecules by either DNA recombination technology or fusion protein technology for integrin targeted therapy have been explored. Furthermore, nanoparticles with integrin targeting ligands for both drug and gene delivery, typically for antiangiogenesis and anticancer therapy, are highlighted and evaluated.What the reader will gain: This review sheds light on the future development of integrin targeted drug and/or gene delivery systems.Take home message: Although thus far there are still limitations, integrin targeted delivery systems have already shown their potential as important pharmaceuticals in the near future.

Absorption, Steady-State Fluorescence, Fluorescence Lifetime, and 2D Self-Assembly Properties of Engineered Fluorescent S-Layer Fusion Proteins of Geobacillus stearothermophilus NRS 2004/3a

S-layer fusion protein technology was used to design four different fluorescent fusion proteins with three different GFP mutants and the red fluorescent protein mRFP1. Their absorption spectra, steady-state fluorescence, and fluorescence lifetime were investigated as a function of pH. It was found that fluorescence intensities and lifetime of the GFP mutant S-layer fusion proteins decreased about 50% between pH 6 and pH 5. The spectral properties of the red S-layer fusion protein were minimally affected by pH variations. These results were compared with His-tagged reference fluorescent proteins, demonstrating that the S-layer protein did not change the general spectral properties of the whole fusion protein. In addition, the pK(a) values of the fluorescent S-layer fusion proteins were calculated. Finally, it was shown that the S-layer fusion proteins were able to self-assemble forming 2D nanostructures of oblique p2 symmetry with lattice parameters of about a = 11 nm, b = 14 nm, and gamma = 80 degrees . The fluorescence tag did not hinder the natural self-assembly process of the S-layer protein. The combination of the fluorescence properties and the self-assembly ability of the engineered fusion proteins make them a promising tool to generate biomimetic surfaces for future applications in nanobiotechnology at a wide range of pH.

Insecticidal activity of scorpion toxin (ButaIT) and snowdrop lectin (GNA) containing fusion proteins towards pest species of different orders

The toxicity of a fusion protein, ButalT/GNA, comprising a venom toxin (ButaIT) derived from the red scorpion, Mesobuthus tamulus (F.), and Galanthus nivalis agglutinin (GNA), was evaluated under laboratory conditions against several pest insects. Insecticidal activity was compared with SFI1/GNA, a fusion comprising a venom toxin (SFI1) derived from the European spider Segestria florentina (Rossi) and GNA, which has been previously demonstrated to be effective against lepidopteran and hemipteran pests, and to GNA itself. Injection assays demonstrated that both fusion proteins were toxic to lepidopteran larvae, dipteran adults, coleopteran adults and larvae and dictyopteran nymphs. ButalT/GNA was more toxic than SFI1/GNA in all cases. GNA itself made a minor contribution to toxicity. Oral toxicity of ButalT/GNA towards lepidopteran pests was confirmed against neonate Spodoptera littoralis (Boisd.), where incorporation at 2% dietary protein resulted in 50% mortality and > 85% reduction in growth compared with controls. ButaIT/GNA was orally toxic to Musca domestica L. adults, causing 75% mortality at 1 mg mL(-1) in aqueous diets and, at 2 mg g(-1) it was orally toxic to Tribolium castaneum (Herbst.), causing 60% mortality and a 90% reduction in growth. Toxicity of the ButaIT/GNA recombinant fusion protein towards a range of insect pests from different orders was demonstrated by injection bioassays. Feeding bioassays demonstrated the potential use of the ButaIT/GNA fusion protein as an orally active insecticide against lepidopteran, dipteran and coleopteran pests. These experiments provide further evidence that the development of fusion protein technology for the generation of new, biorational, anti-insect molecules holds significant promise.

Co-localisation studies of Arabidopsis SR splicing factors reveal different types of speckles in plant cell nuclei

SR proteins are multidomain splicing factors which are important for spliceosome assembly and for regulation of alternative splicing. In mammalian nuclei these proteins localise to speckles from where they are recruited to transcription sites. By using fluorescent protein fusion technology and different experimental approaches it has been shown that Arabidopsis SR proteins, in addition to diffuse nucleoplasmic staining, localise into an irregular nucleoplasmic network resembling speckles in mammalian cells. As Arabidopsis SR proteins fall into seven conserved sub-families we investigated co-localisation of members of the different sub-families in transiently transformed tobacco protoplast. Here we demonstrate the new finding that members of different SR protein sub-families localise into distinct populations of nuclear speckles with no, partial or complete co-localisation. This is particularly interesting as we also show that these proteins do interact in a yeast two-hybrid assay as well as in pull-down and in co-immunopreciptiation assays. Our data raise the interesting possibility that SR proteins are partitioned into distinct populations of nuclear speckles to allow a more specific recruitment to the transcription/pre-mRNA processing sites of particular genes depending on cell type and developmental stage.

Production of active eukaryotic proteins through bacterial expression systems: a review of the existing biotechnology strategies

Among the various expression systems employed for the over-production of proteins, bacteria still remains the favorite choice of a Protein Biochemist. However, even today, due to the lack of post-translational modification machinery in bacteria, recombinant eukaryotic protein production poses an immense challenge, which invariably leads to the production of biologically in-active protein in this host. A number of techniques are cited in the literature, which describe the conversion of inactive protein, expressed as an insoluble fraction, into a soluble and active form. Overall, we have divided these methods into three major groups: Group-I, where the factors influencing the formation of insoluble fraction are modified through a stringent control of the cellular milieu, thereby leading to the expression of recombinant protein as soluble moiety; Group-II, where protein is refolded from the inclusion bodies and thereby target protein modification is avoided; Group-III, where the target protein is engineered to achieve soluble expression through fusion protein technology. Even within the same family of proteins (e.g., tyrosine kinases), optimization of standard operating protocol (SOP) may still be required for each protein's over-production at a pilot-scale in Escherichia coli. However, once standardized, this procedure can be made amenable to the industrial production for that particular protein with minimum alterations.

Drug Targeting to the Brain

The goal of brain drug targeting technology is the delivery of therapeutics across the blood-brain barrier (BBB), including the human BBB. This is accomplished by re-engineering pharmaceuticals to cross the BBB via specific endogenous transporters localized within the brain capillary endothelium. Certain endogenous peptides, such as insulin or transferrin, undergo receptor-mediated transport (RMT) across the BBB in vivo. In addition, peptidomimetic monoclonal antibodies (MAb) may also cross the BBB via RMT on the endogenous transporters. The MAb may be used as a molecular Trojan horse to ferry across the BBB large molecule pharmaceuticals, including recombinant proteins, antibodies, RNA interference drugs, or non-viral gene medicines. Fusion proteins of the molecular Trojan horse and either neurotrophins or single chain Fv antibodies have been genetically engineered. The fusion proteins retain bi-functional properties, and both bind the BBB receptor, to trigger transport into brain, and bind the cognate receptor inside brain to induce the pharmacologic effect. Trojan horse liposome technology enables the brain targeting of non-viral plasmid DNA. Molecular Trojan horses may be formulated with fusion protein technology, avidin-biotin technology, or Trojan horse liposomes to target to brain virtually any large molecule pharmaceutical.

A fused selenium-containing protein with both GPx and SOD activities

As a safeguard against oxidative stress, the balance between the main antioxidant enzymes including superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) was believed to be more important than any single one, for example, dual-functional SOD/CAT enzyme has been proved to have better antioxidant ability than either single enzyme. By combining traditional fusion protein technology with amino acid auxotrophic expression system, we generated a bifunctional enzyme with both GPx and SOD activities. It displayed better antioxidant ability than GPx or SOD. Such dual-functional enzymes could facilitate further studies of the cooperation of GPx and SOD and generation of better therapeutic agents.

Recombinant granulocyte colony-stimulating factor-transferrin fusion protein as an oral myelopoietic agent

An expression construct harboring granulocyte colony-stimulating factor (G-CSF)-transferrin (Tf) fusion protein (G-CSF-Tf) was engineered by fusing human cDNAs encoding G-CSF and Tf to explore the feasibility of using Tf as a carrier moiety for oral delivery of therapeutic proteins. The recombinant protein, G-CSF-Tf, was harvested from protein-free, conditioned medium of transfected HEK293 cells. The in vitro studies demonstrated that the purified G-CSF-Tf fusion protein possesses the activity of both Tf receptor (TfR) binding in Caco-2 cells and G-CSF-dependent stimulation of NFS-60 cell proliferation. Subcutaneous administration of G-CSF-Tf fusion protein to BDF1 mice demonstrated a pharmacological effect comparable to the commercial G-CSF on the increase of absolute neutrophil counts (ANC). However, the fusion protein elicited a significant increase in ANC upon oral administration to BDF1 mice, whereas G-CSF had no effect. This study also showed that orally administered G-CSF-Tf elicits a sustained myelopoietic effect up to 3 days, whereas the s.c. administered G-CSF or G-CSF-Tf lasts only 1 day. Furthermore, coadministration of free Tf abolished the increase of ANC by orally delivered G-CSF-Tf, suggesting that the recombinant protein is absorbed via a TfR-mediated process in the gastrointestinal tract. Taken together, we conclude that the Tf-based recombinant fusion protein technology represents a promising approach for future development of orally effective peptide and protein drugs.

Dynamic localization and persistent stimulation of factor-dependent cells by a stem cell factor / cellulose binding domain fusion protein

The extracellular matrix provides structural components that support the development of tissue morphology and the distribution of growth factors that modulate the overall cellular response to those growth factors. The ability to manipulate the presentation of factors in culture systems should provide an additional degree of control in regulating the stimulation of factor-dependent cells for tissue engineering applications. Cellulose binding domain (CBD) fusion protein technology facilitates the binding of bioactive cytokines to cellulose materials, and has permitted the analysis of several aspects of cell stimulation by surface-localized growth factors. We previously reported the synthesis and initial characterization of a fusion protein comprised of a CBD and murine stem cell factor (SCF) (Doheny et al. [1999] Biochem J 339:429-434). A significant advantage of the CBD fusion protein system is that it permits the stimulation of factor-dependent cells with localized growth factor, essentially free of nonfactor-derived interactions between the cell and matrix. In this work, the long-term stability and bioactivity of SCF-CBD fusions adsorbed to microcrystalline cellulose under cell culture conditions is demonstrated. Cellulose-bound SCF-CBD is shown to stimulate receptor polarization in the cell membrane and adherence to the cellulose matrix. In addition, cellulose-surface presentation of the SCF-CBD attenuates c-kit dephosphorylation kinetics, potentially modulating the overall response of the cell to the SCF signal.

Advanced genetic strategies for recombinant protein expression in Escherichia coli

Preparations enriched by a specific protein are rarely easily obtained from natural host cells. Hence, recombinant protein production is frequently the sole applicable procedure. The ribosomal machinery, located in the cytoplasm is an outstanding catalyst of recombinant protein biosynthesis. Escherichia coli facilitates protein expression by its relative simplicity, its inexpensive and fast high-density cultivation, the well-known genetics and the large number of compatible tools available for biotechnology. Especially the variety of available plasmids, recombinant fusion partners and mutant strains have advanced the possibilities with E. coli. Although often simple for soluble proteins, major obstacles are encountered in the expression of many heterologous proteins and proteins lacking relevant interaction partners in the E. coli cytoplasm. Here we review the current most important strategies for recombinant expression in E. coli. Issues addressed include expression systems in general, selection of host strain, mRNA stability, codon bias, inclusion body formation and prevention, fusion protein technology and site-specific proteolysis, compartment directed secretion and finally co-overexpression technology. The macromolecular background for a variety of obstacles and genetic state-of-the-art solutions are presented.

Transduction of functionally active TAT fusion proteins into cornea

A technology has recently been developed that allows for the rapid transduction of full-length functionally active proteins into intact tissue through intravenous injection and into cultured cells. This technology involves the fusion of an 11 amino acid sequence of the HIV TAT protein to the protein of interest. In the current investigation, we determined whether functionally active TAT fusion proteins could be transduced into intact corneas by topical application. TAT-β-galactosidase was purified from bacterial cells and applied in serial dilutions (12·5–250 n m) to cultured epithelial cells for 5 or 15 min. In addition, enucleated globes and excised corneas with or without a central 3-mm epithelial debridement were incubated with TAT-β-galactosidase for 1 or 2 hr. Excised corneas were allowed to heal in organ culture. Transduction of active β-galactosidase was detected by incubating the cells or corneas with X-gal. TAT-β-galactosidase was transduced into nearly all cultured cells in a concentration-dependent manner. When TAT-β-galactosidase was topically applied to intact corneas, only the most superficial layer of epithelium was highly transduced. When the superficial layer was removed with nitrocellulose, two to four layers of cells were transduced. In corneas with a central debridement, epithelial cells at the edge of the debridement were transduced as well as the stromal cells subjacent to the debridement. Active β-galactosidase was maintained at least 1 day in organ culture. No X-gal reaction was seen in either cells or corneas not incubated with TAT-β-galactosidase. Functionally active proteins can be efficiently transduced into corneal epithelial and stromal cells using TAT fusion protein technology. The intact epithelium provides a barrier to penetration of TAT proteins. This barrier can be overcome by disrupting the epithelium. TAT-mediated protein transduction may be extremely useful in studies of corneal wound healing and homeostasis.

Generation of low-toxicity interleukin-2 fusion proteins devoid of vasopermeability activity

AbstractBecause of its key role in immunity, interleukin-2 (IL-2) has been studied extensively for the adoptive immunotherapy of cancer. Although systemic administration of IL-2 has been shown to stimulate antitumor responses in vivo, its efficacy in the clinic has been limited by the development of serious side effects, including the induction of vascular leak syndrome. Previously, we have identified a small peptide fragment of IL-2 that was found to contain the entire vasopermeability activity of the cytokine. The identification of the location of this potentially undesirable property of IL-2 enabled us to focus on the generation of mutant derivatives that might be lacking vasopermeability activity but that retain cytokine functionality. In addition to this discovery, our laboratory has constructed monoclonal antibody/IL-2 fusion proteins that can target this potent cytokine directly to tumor for the immunotherapy of both solid and lymphoid malignancies. Using this fusion protein technology, we have constructed a series of point mutations in the newly identified vasopermeability region of IL-2 for the purpose of deleting this activity. Fusion proteins showing reduced or deleted vasopermeability activity were then tested for their cytokine potency by several methods, including their binding to IL-2 receptors, T-cell proliferation assays, the induction of secondary cytokines, dose-escalating toxicity, and finally their ability to treat established solid tumors in syngeneic immunocompetent mice. The results of these studies clearly show that the vasopermeability activity of IL-2 can be substantially deleted by single point mutations such as Arg38Trp without grossly affecting the immune function of the cytokine.

Induction of antigen-specific CTL by recombinant HIV trans-activating fusion protein-pulsed human monocyte-derived dendritic cells.

Several systems have been tested for introduction of Ags into human dendritic cells (DC). Most of them to date, however, are complex and possess limited efficiency. Recent advances in HIV trans-activating (TAT) fusion protein technology permit extremely high transduction efficiencies for a majority of mammalian cell types. Here we report our attempts to develop a simple, but highly efficient, protocol for loading of antigenic protein into DC using TAT fusion technology. A TAT-minigene fusion protein was generated, encoding both the HLA-A2-restricted influenza matrix protein-derived epitope (GILVFTFTL, Flu-M1) and a melanoma Ag gp100-derived modified epitope (YLEPGPVTV, G9(280)-9V). In addition, both a TAT-Her2/neu extracellular domain (ECD) fusion protein and a TAT-green fluorescence protein fusion protein were generated. Over 95% of DC stained positively for TAT-green fluorescence protein within 20 min of coculture. DC treated with TAT-minigene were efficiently recognized by both Flu-M1 and G9(280)-9V-specific T cells in cytotoxicity assays and IFN-gamma ELISPOT assays. In contrast, DC pulsed with minigene fusion protein lacking TAT were either poorly recognized or not recognized by the T cells. DC pulsed with TAT-minigene also efficiently induced Flu-M1-specific T cells from naive lymphocytes. Similarly, DC treated with TAT-Her2/neu ECD stimulated patient-derived lymphocytes that specifically recognized Her2/neu(+) ovarian and breast cancer cell lines. The CTL induced by TAT-Her2/neu ECD-pulsed DC specifically recognized the Her2/neu ECD-derived immunogenic peptide E75 (KIFGSLAFL). Our data suggest that TAT fusion proteins efficiently transduce DC and induce Ag-specific T cells. This could prove to be a useful method for treatment of infectious diseases and cancer.

A fluorescent cassette-based strategy for engineering multiple domain fusion proteins

BackgroundThe engineering of fusion proteins has become increasingly important and most recently has formed the basis of many biosensors, protein purification systems, and classes of new drugs. Currently, most fusion proteins consist of three or fewer domains, however, more sophisticated designs could easily involve three or more domains. Using traditional subcloning strategies, this requires micromanagement of restriction enzymes sites that results in complex workaround solutions, if any at all.ResultsTherefore, to aid in the efficient construction of fusion proteins involving multiple domains, we have created a new expression vector that allows us to rapidly generate a library of cassettes. Cassettes have a standard vector structure based on four specific restriction endonuclease sites and using a subtle property of blunt or compatible cohesive end restriction enzymes, they can be fused in any order and number of times. Furthermore, the insertion of PCR products into our expression vector or the recombination of cassettes can be dramatically simplified by screening for the presence or absence of fluorescence.ConclusionsFinally, the utility of this new strategy was demonstrated by the creation of basic cassettes for protein targeting to subcellular organelles and for protein purification using multiple affinity tags.

Recombinant enterokinase light chain with affinity tag: expression from Saccharomyces cerevisiae and its utilities in fusion protein technology.

Enterokinase and recombinant enterokinase light chain (rEK(L)) have been used widely to cleave fusion proteins with the target sequence of (Asp)(4)-Lys. In this work, we show that their utility as a site-specific cleavage agent is compromised by sporadic cleavage at other sites, albeit at low levels. Further degradation of the fusion protein in cleavage reaction is due to an intrinsic broad specificity of the enzyme rather than to the presence of contaminating proteases. To offer facilitated purification from fermentation broth and efficient removal of rEK(L) after cleavage reaction, thus minimizing unwanted cleavage of target protein, histidine affinity tag was introduced into rEK(L). Utilizing the secretion enhancer peptide derived from the human interleukin 1 beta, the recombinant EK(L) was expressed in Saccharomyces cerevisiae and efficiently secreted into culture medium. The C-terminal His-tagged EK(L) was purified in a single-step procedure on nickel affinity chromatography. It retained full enzymatic activity similar to that of EK(L), whereas the N-terminal His-tagged EK(L) was neither efficiently purified nor had any enzymatic activity. After cleavage reaction of fusion protein, the C-terminal His-tagged EK(L) was efficiently removed from the reaction mixture by a single passage through nickel-NTA spin column. The simple affinity tag renders rEK(L) extremely useful for purification, post-cleavage removal, recovery, and recycling and will broaden the utility and the versatility of the enterokinase for the production of recombinant proteins.

A somatic gene transfer approach using recombinant fusion proteins to map muscle-motoneuron projections in Xenopus spinal cord.

A combination of somatic gene transfer with fusion protein technology has been developed, thus providing an innovative means of mapping muscle-motoneuronal connections in Xenopus tadpole spinal cord. We analyzed whether a neuronal tracer created by the fusion of the LacZ gene to the tetanus toxin C fragment (LacZ-TTC) could be produced from plasmid DNA injected into muscle, and whether it could be released and undergo retrograde transport into motoneurons. Plasmids encoding various fusion protein constructions, with or without a signal peptide, were injected into dorsal or caudal muscles of premetamorphic tadpoles. The marker was produced in the muscle at constantly high levels. At one month post-injection, the fusion protein passed the neuromuscular junction and underwent retrograde transport into motoneurons. Transfer into motoneurons was seen for every animal injected, emphasizing the high reproducibility and efficiency of the process. No uptake of beta-gal protein into motoneurons was observed in the absence of the TTC fragment. Furthermore, no enhancement was obtained by adding a signal peptide. These results provide the first demonstration of the synthesis and transport of a TTC fusion protein produced directly from exogenous DNA in a vertebrate system.

Determinants of Recombinant Production of Antimicrobial Cationic Peptides and Creation of Peptide Variants in Bacteria

Cationic peptides possessing antibacterial activity are virtually ubiquitous in nature, and offer exciting prospects as new therapeutic agents. We had previously demonstrated that such peptides could be produced by fusion protein technology in bacteria and several carrier proteins had been tested as fusion partners including glutathione-S-transferase, S. aureusprotein A, IgG binding protein and P. aeruginosaouter membrane protein OprF. However these fusion partners, while successfully employed in peptide expression, were not optimized for high level production of cationic peptides (Piers, K., Brow, M. L., and Hancock, R. E. W. 1993, Gene137, 7–13). In this paper we took advantage of a small replication protein RepA from E. coliand used its truncated version to construct fusion partners. The minimal elements required for high level expression of cationic peptide were defined as a DNA sequence encoding a fusion protein comprising, from the N-terminus, a 68 amino acid carrier region, an anionic prepro domain, a single methionine and the peptide of interest. The 68 amino acid carrier region was a block of three polypeptides consisting of a truncated RepA, a synthetic cellulose binding domain and a hexa histidine domain. The improved system showed high level expression and simplified downstream purification. The active peptide could be yielded by CNBr cleavage of the fusion protein. This novel vector was used to express three classes of cationic peptides including the α-helical peptide CEMA, the looped peptide bactenecin and the extended peptide indolicidin. In addition, mutagenesis of the peptide gene to produce peptide variants of CEMA and indolicidin using the improved vector system was shown to be successful.

Beta PDGFR-IgG chimera demonstrates that human beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding.

To localize human beta PDGFR binding determinants, we constructed a fusion protein comprising beta PDGFR Ig-like domains 1 to 3 and an IgG1 Fc domain (beta PDGFR-HFc). beta PDGFR-HFc was expressed as a 200 kDa dimeric molecule and contained Fc epitopes as demonstrated by anti-mouse Fc antibody recognition. Scatchard analysis revealed that PDGF BB possessed a dissociation constant of 1.5 nM for beta PDGFR-HFc. Thus, beta PDGFR Ig-like domains 1 to 3 are sufficient for high affinity PDGF BB binding. We exploited this fusion protein technology to identify and characterize beta PDGFR antagonists using a sensitive beta PDGFR immunosorbent assay. In this assay, beta PDGFR-HFc half-maximally bound to PDGF BB with an affinity of around 150 pM. Suramin, as well as bacterially expressed and refolded human alpha PDGFR domains 1-3, inhibited beta PDGFR-HFc binding to PDGF BB half-maximally at 25 microM and 10 nM respectively. Therefore, alpha PDGFR D1-3, like beta PDGFR D1-3, are sufficient for high affinity PDGF BB binding. Furthermore, the beta PDGFR-HFc immunosorbent assay will be useful to identify beta PDGFR antagonists as well as to study alpha and beta PDGFR substitution mutants which further map receptor binding determinants.

N2,N2-dimethylguanosine-specific tRNA methyltransferase contains both nuclear and mitochondrial targeting signals in Saccharomyces cerevisiae.

The TRM1 gene of Saccharomyces cerevisiae encodes a tRNA modification enzyme, N2,N2-dimethylguanosine-specific tRNA methyltransferase, which modifies both mitochondrial and cytoplasmic tRNAs. The enzyme is targeted to mitochondria for the modification of mitochondrial tRNAs. Cellular fractionation and indirect immunofluorescence studies reported here demonstrate that this enzyme is also localized to the nucleus. Further, immunofluorescence experiments using strains that overproduce the enzyme show a staining at the periphery of the nucleus suggesting that the enzyme is found in a subnuclear destination near or at the nuclear membrane. There is no obvious cytoplasmic staining in these overproducing strains. Fusion protein technology was used to begin to localize sequences involved in the nuclear targeting of this enzyme. Indirect immunofluorescence studies indicate that sequences between the first 70 and 213 NH2-terminal amino acids of the methyltransferase are sufficient to target Escherichia coli beta-galactosidase to nuclei.