Large-scale Production And Purification Of Recombinant Protein From An Insect Cell/baculovirus System In Erlenmeyer Flasks: Application To The Chicken Poly(adp-ribose) Polymerase Catalytic Domain

A simple and inexpensive shaker/Erlenmeyer flask system for large-scale cultivation of insect cells is described and compared to a commercial spinner system. On the basis of maximum cell density, average population doubling time and overproduction of recombinant protein, a better result was obtained with a simpler and less expensive bioreactor consisting of Erlenmeyer flasks and an ordinary shaker waterbath. Routinely, about 90 mg of pure poly(ADP-ribose) polymerase catalytic domain was obtained for a total of 3 × 109 infected cells in three liters of culture.308923928Althaus, F.R., Richter, C., ADP-ribosylation of proteins. Enzymology and biological significance (1987) Molecular Biology, Biochemistry and Biophysics, 37, pp. 1-126Lautier, D., Lagueaux, J., Ménard, L., Poirier, G.C., Molecular and biochemical features of poly(ADP-ribose) metabolism (1993) Molecular and Cellular Biochemistry, 122, pp. 171-193Satoh, M.S., Lindahl, T., Role of poly(ADP-ribose) formation in DNA repair (1992) Nature, 356, pp. 356-358Molinete, M., Vermeulen, W., Bürkle, A., Ménissier-de Murcia, J., Küpper, J., Hoeijmakers, J., De Murcia, G., Overproduction of the poly(ADP-ribose) polymerase DNA-binding domain blocks alkylation-induced DNA repair synthesis in mammalian cells (1993) EMBO Journal, 5, pp. 2109-2117Masson, M., Rolli, V., Dantzer, F., Trucco, C., Schrieber, V., Fribourg, S., Molinete, M., De Murcia, G., Poly(ADP-ribose) polymerase: Structure-function relationship (1995) Biochimie, 77, pp. 456-461Suto, M.L., Suto, M.S., Inhibitors of poly(ADP-ribose) polymerase (ADPRP): Potential chemotherapeutic agents (1991) Drugs of the Future, 16, pp. 723-739Giner, H., Simonin, F., De Murcia, G., Ménissier-de Murcia, J., Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system (1992) Gene, 114, pp. 279-283Simonin, F., Höfferer, L., Panzeter, P., Muller, S., De Murcia, G., Althaus, F.R., The carboxy-terminal domain of human poly(ADP-ribose) polymerase: Overproduction in Escherichia coli, large scale purification and characterization (1993) Journal of Molecular Biology, 268, pp. 13454-13461Murhammer, D.W., Review and patents literature. The use of insect cell cultures for recombinant protein synthesis: Engineering aspects (1991) Applied Biochemistry and Biotechnology, 31, pp. 283-310Goosen, M.F.A., Large-scale insect cell culture (1992) Current Opinion in Biotechnology, 3, pp. 99-104Kamen, A.A., Tom, R.L., Caron, A.W., Chavarie, C., Massie, B., Archambault, J., Culture of insect cells in a helical ribbon impeller bioreactor (1991) Biotechnology and Bioengineering, 38, pp. 619-628Van Lier, F.L.J., Van Den End, E.J., Gooijer, C.D., Vlak, J.M., Tramper, J., Continuous production of baculovirus in a cascade insect-cell reactor (1990) Applied Microbiology and Biotechnology, 33, pp. 43-47Power, J., Greenfield, P.F., Nielsen, L., Reid, S., Modelling the growth and protein production by insect cells following infection by a recombinant baculovirus in suspension culture (1992) Cytotechnology, 9, pp. 149-155Stavroulakis, D.A., Kalogerakis, N., Behie, L.A., Latrou, K., Kinetic data for the BM-5 insect line in repeated-batch suspension culture (1991) Biotechnology, 38, pp. 116-126Neutra, R., Levi, B.-Z., Shoham, Y., Optimization of protein-production by the baculovirus expression vector system in shake flasks (1992) Applied Microbiology and Biotechnology, 37, pp. 74-78Luckow, V.A., Summers, M.D., Signals important for high level expression of foreign genes in Autographa californica nuclear polyhedrosis virus expression vectors (1989) Virology, 170, pp. 31-39O'Reily, D.R., Miller, L.K., Luckow, V.A., (1992) Baculovirus Expression System: A Laboratory Manual, , Freeman, New YorkScott, R.L., Blanchard, J.H., Fergusson, C.H.R., Effects of oxygen on recombinant protein production by suspension cultures of Spodoptera frugiperda Sf9 insect cell (1992) Enzyme and Microbial Technology, 14, pp. 798-80

The BRCT Domain of PARP-1 Is Required for Immunoglobulin Gene Conversion

During affinity maturation, genomic integrity is maintained through specific targeting of DNA mutations. The DNA damage sensor PARP-1 helps determine whether a DNA lesion results in faithful or mutagenic repair

PolyADP-Ribosylation Is Required for Pronuclear Fusion during Postfertilization in Mice

BACKGROUND: During fertilization, pronuclear envelope breakdown (PNEB) is followed by the mingling of male and female genomes. Dynamic chromatin and protein rearrangements require posttranslational modification (PTM) for the postfertilization development. METHODOLOGY/PRINCIPAL FINDINGS: Inhibition of poly(ADP-ribose) polymerase activity (PARylation) by either PJ-34 or 5-AIQ resulted in developmental arrest of fertilized embryos at the PNEB. PARylation inhibition affects spindle bundle formation and phosphorylation of Erk molecules of metaphase II (MII) unfertilized oocytes. We found a frequent appearance of multiple pronuclei (PN) in the PARylation-inhibited embryos, suggesting defective polymerization of tubulins. Attenuated phosphorylation of lamin A/C by PARylation was detected in the PARylation-inhibited embryos at PNEB. This was associated with sustained localization of heterodomain protein 1 (HP1) at the PN of the one-cell embryos arrested by PARylation inhibition. CONCLUSIONS/SIGNIFICANCE: Our findings indicate that PARylation is required for pronuclear fusion during postfertilization processes. These data further suggest that PARylation regulates protein dynamics essential for the beginning of mouse zygotic development. PARylation and its involving signal-pathways may represent potential targets as contraceptives

Targeting poly(ADP-ribose) polymerase activity for cancer therapy

Poly(ADP-ribosyl)ation is a ubiquitous protein modification found in mammalian cells that modulates many cellular responses, including DNA repair. The poly(ADP-ribose) polymerase (PARP) family catalyze the formation and addition onto proteins of negatively charged ADP-ribose polymers synthesized from NAD+. The absence of PARP-1 and PARP-2, both of which are activated by DNA damage, results in hypersensitivity to ionizing radiation and alkylating agents. PARP inhibitors that compete with NAD+ at the enzyme’s activity site are effective chemo- and radiopotentiation agents and, in BRCA-deficient tumors, can be used as single-agent therapies acting through the principle of synthetic lethality. Through extensive drug-development programs, third-generation inhibitors have now entered clinical trials and are showing great promise. However, both PARP-1 and PARP-2 are not only involved in DNA repair but also in transcription regulation, chromatin modification, and cellular homeostasis. The impact on these processes of PARP inhibition on long-term therapeutic responses needs to be investigated

Poly(ADP-ribose) polymerase: molecular biological aspects

A number of roles have been ascribed to poly(ADP-ribose) polymerase* including involvement in DNA repair, cell proliferation, differentiation and transformation. Cloning of the gene has allowed the development of molecular biological approaches to elucidate the structure and the function(s) of this highly conserved enzyme. This article will review the recent results obtained in this field

Random mutagenesis of the poly(ADP-ribose) polymerase catalytic domain reveals amino acids involved in polymer branching

Poly(ADP-ribose) polymerase (PARP) is a multifunctional nuclear zinc finger protein which participates in the immediate response of mammalian cells exposed to DNA damaging agents. Given the complexity of the poly(ADP-ribosylation) reaction, we developed a large-scale screening procedure in Escherichia coli to identify randomly amino acids involved in the various aspects of this mechanism. Random mutations were generated by the polymerase chain reaction in a cDNA sequence covering most of the catalytic domain. Out of 26 individual mutations that diversely inactivated the full-length PARP, 22 were found at conserved positions in the primary structure, and 24 were located in the core domain formed by two beta-sheets containing the active site. Most of the PARP mutants were altered in poly(ADP-ribose) elongation and/or branching. The spatial proximity of some residues involved in chain elongation (E988) and branching (Y986) suggests a proximity or a superposition of these two catalytic sites. Other residues affected in branching were located at the surface of the molecule (R847, E923, G972), indicating that protein-protein contacts are necessary for optimal polymer branching. This screening procedure provides a simple and efficient method to explore further the structure-function relationship of the enzyme

Overproduction and large-scale purification of the human poly(ADP-ribose) polymerase using a baculovirus expression system

We have overproduced the full-length human poly(ADP-ribose) polymerase (PARP) in Spodoptera frugiperda (Sf9) cells using a baculovirus expression vector system. Approx. 20 mg of purified protein from 5 x 10(8) Sf9 cells were obtained by a simple three-step purification procedure including 3-aminobenzamide affinity chromatography. The recombinant protein (rePARP), which migrates as a unique 116-kDa band on SDS-polyacrylamide gels, was identified as PARP by Western blotting using either polyclonal or monoclonal antibodies raised against the purified human and calf thymus enzymes. Furthermore, rePARP is a functional protein, as demonstrated by its ability to specifically bind Zn2+ and DNA, and to recognize single-strand breaks in DNA. The purified enzyme has the same affinity for NAD+ and turnover number as the human placental PARP. Thus, rePARP produced in insect cells is biologically active and suitable for functional analysis. The reproducibility of the overproduction and the simplicity of the purification protocol, as well as the yield of the produced protein, should greatly facilitate physicochemical and structural studies

Electron microscopic mapping of single-stranded discontinuities in cauliflower mosaic virus DNA by means of the biotin-avidin technique

Biotin-labeled deoxyuridine triphosphate has been used to label the three natural single-stranded discontinuities of Cauliflower Mosaic Virus DNA. The presence of biotin permitted the direct visualization and mapping of the discontinuities by electron microscopy, using ferritin-labeled avidin