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

Miranda, Everson Alves; DeMurcia, G.; Ménissier-de-Murcia, J.

doi:10.1590/s0100-879x1997000800002

Cited by 7 publications

(6 citation statements)

References 14 publications

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“…The advantages with the baculovirus system are that insect cells can be grown in large scale in suspension cultures and without serum (47). We found, however, that the expression level of LPL was low compared with other lipases that have been expressed by this method (27,48).…”

Section: Table II Effect Of Co-expression With Crt On Lpl Dimermentioning

confidence: 84%

Calreticulin Promotes Folding/Dimerization of Human Lipoprotein Lipase Expressed in Insect Cells (Sf21)

Zhang

Tate

et al. 2003

Journal of Biological Chemistry

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Lipoprotein lipase (LPL) is a non-covalent, homodimeric, N-glycosylated enzyme important for metabolism of blood lipids. LPL is regulated by yet unknown post-translational events affecting the levels of active dimers. On co-expression of LPL with human molecular chaperones, we found that calreticulin had the most pronounced effects on LPL activity, but calnexin was also effective. Calreticulin caused a 9-fold increase in active LPL, amounting to about 50% of the expressed LPL protein. The total expression of LPL protein was increased less than 20%, and the secretion rates for active and inactive LPL were not significantly changed by the chaperone. Thus, the main effect was an increased specific activity of LPL both in cells and media. Chromatography on heparin-Sepharose and sucrose density gradient centrifugation demonstrated that most of the inactive LPL was monomeric and that calreticulin promoted formation of active dimers. Higher oligomers of inactive LPL were present in cell extracts, but only monomers and dimers were secreted to the medium. Interaction between LPL and calreticulin was demonstrated, and the effect of the chaperone was prevented by castanospermine, an inhibitor of N-glycan glucose trimming. Our data indicate an important role of endoplasmic reticulum-based chaperones for the folding/ dimerization of LPL. Lipoprotein lipase (LPL)1 hydrolyzes triglycerides in the circulating plasma lipoproteins, such as chylomicrons and very low density lipoproteins (1-3). The enzyme is produced and secreted mainly by adipocytes and muscle cells, but it is also produced in other cells such as macrophages and certain neurons. LPL is transported by yet unclear mechanisms to the vascular endothelium, where it is attached to cell surfaces through interaction with heparan sulfate proteoglycans (HSPG). The products of lipolysis are taken up for storage or oxidation in adjacent tissues. In addition, LPL can mediate internalization of intact lipoproteins by bridging them to cell surface receptors like the LDL receptor-like protein or HSPG.LPL, along with pancreatic lipase, hepatic lipase (HL), and endothelial lipase, belongs to the gene family of mammalian triglyceride lipases (2, 4). The three-dimensional structure of LPL has not yet been determined. Based on homology with pancreatic lipase, it has been proposed that LPL has two structurally distinct regions, a larger N-terminal domain containing the active site and a smaller C-terminal domain with other important functions, such as binding to lipids, LDL receptorlike protein, and HSPG (5, 6).Catalytically active LPL is a non-covalent, homodimeric glycoprotein (7, 8). In the rough endoplasmic reticulum (ER), human LPL is co-translationally glycosylated at amino acid residues Asn 43 and Asn 359 . Studies using site-directed mutagenesis and inhibitors of transport between ER and Golgi have shown that core glycosylation at residue Asn 43 is necessary for activation and release of LPL from the ER, but glycosylation at Asn 359 is not necessary (9 -11). A number of studies...

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Section: Table II Effect Of Co-expression With Crt On Lpl Dimermentioning

confidence: 84%

Calreticulin Promotes Folding/Dimerization of Human Lipoprotein Lipase Expressed in Insect Cells (Sf21)

Zhang

Tate

et al. 2003

Journal of Biological Chemistry

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“…Cell propagation and protein production was performed as described previously (22). The identity of the purified protein was confirmed by Western blot with the polyclonal antibody against PARP-2 (1:2000 dilution).…”

Section: Est Searches and Cloning Of Cdna Encoding Murine Parp-2-mentioning

confidence: 99%

PARP-2, A Novel Mammalian DNA Damage-dependent Poly(ADP-ribose) Polymerase

Amé¹,

Rolli²,

Schreiber³

et al. 1999

Journal of Biological Chemistry

653

311

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Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins in response to DNA damage that activates the base excision repair machinery. Poly-(ADP-ribose) polymerase which we will now call PARP-1, has been the only known enzyme of this type for over 30 years. Here, we describe a cDNA encoding a 62-kDa protein that shares considerable homology with the catalytic domain of PARP-1 and also contains a basic DNA-binding domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and 14q11.2 in mouse and human, respectively. Purified recombinant mouse PARP-2 is a damaged DNA-binding protein in vitro and catalyzes the formation of poly(ADP-ribose) polymers in a DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleus in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.In response to DNA-strand breaks introduced either directly by ionizing radiation or indirectly following enzymatic incision at a DNA lesion, the immediate poly(ADP-ribosylation) of nuclear proteins converts the DNA ends into intracellular signals that modulate DNA repair and cell survival programs. At the sites of DNA breakage, poly(ADP-ribose) polymerase (PARP) 1 (EC 2.4.2.30) catalyzes the transfer of the ADP-ribose moiety from its substrate NAD ϩ , to a limited number of proteins involved in chromatin architecture, DNA repair, or in DNA metabolism including PARP itself (1-4). Recently, the generation of PARP-deficient mice by homologous recombination (5, 6) has clearly demonstrated the involvement of PARP in the maintenance of the genomic integrity due to its role during base excision repair (7-9). An substantial delay in DNA strandbreak repair was observed following treatment of PARP-deficient cells with monofunctional alkylating agents (10). This severe DNA repair defect appears to be the primary cause for the observed cytotoxicity of N-methyl-N-nitrosourea, methylmethanesulfonate (MMS), or ␥-rays leading to cell death occurring after a G 2 /M block (10).It was assumed for many years that PARP activity was associated with a single protein displaying unique DNA damage detection and signaling properties. This assumption was challenged by the recent discovery in Arabidopsis thaliana of a gene coding for a PARP-related polypeptide of a calculated molecular mass of 72 kDa (11). It then became evident that two structurally different PARP proteins, both possessing DNA-dependent poly(ADP-ribose) activities, were present in both A. thaliana as well as in maize (12)(13)(14). 2 Furthermore, it has been reported recently that mouse embryonic fibroblasts derived from PARP knockout are capable of synthesizing ADP-ribose polymers in response to DNA damage (15), suggesting that in mammals, like in plants, at least one additional member of the PARP family may exist in addition to the classical zinc finger containing PAR...

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“…Sf9 cell propagation and protein production was performed according to Miranda et al (Miranda et al, 1997). Purification of hPARP-3 was performed by affinity chromatography on Affigel-3-Aminobenzamide as previously described (Amé et al, 1999;Giner et al, 1992).…”

Section: Fluorescence In Situ Hybridization (Fish) Analysismentioning

confidence: 99%

PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression

Augustin

Spenlehauer

Dumond

et al. 2003

Journal of Cell Science

136

115

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A novel member of the poly(ADP-ribose) polymerase (PARP) family, hPARP-3,is identified here as a core component of the centrosome. hPARP-3 is preferentially localized to the daughter centriole throughout the cell cycle. The N-terminal domain (54 amino acids) of hPARP-3 is responsible for its centrosomal localization. Full-length hPAPR-3 (540 amino acids, with an apparent mass of 67 kDa) synthesizes ADP-ribose polymers during its automodification. Overexpression of hPARP-3 or its N-terminal domain does not influence centrosomal duplication or amplification but interferes with the G1/S cell cycle progression. PARP-1 also resides for part of the cell cycle in the centrosome and interacts with hPARP-3. The presence of both PARP-1 and PARP-3 at the centrosome may link the DNA damage surveillance network to the mitotic fidelity checkpoint.

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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

Cited by 7 publications

References 14 publications

Calreticulin Promotes Folding/Dimerization of Human Lipoprotein Lipase Expressed in Insect Cells (Sf21)

Calreticulin Promotes Folding/Dimerization of Human Lipoprotein Lipase Expressed in Insect Cells (Sf21)

PARP-2, A Novel Mammalian DNA Damage-dependent Poly(ADP-ribose) Polymerase

PARP-3 localizes preferentially to the daughter centriole and interferes with the G1/S cell cycle progression

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