Active Recombinant Rat Dipeptidyl Aminopeptidase I (Cathepsin C) Produced Using the Baculovirus Expression System

Lauritzen, Conni; Pedersen, John; Madsen, Mads Thorup; Justesen, Just; Martensen, Pia M.; Dahl, Søren

doi:10.1006/prep.1998.0976

Cited by 32 publications

(26 citation statements)

References 30 publications

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“…We used shaker culture at the 8-to 12-L scale to obtain sufficient quantities of protein, an approach that has several advantages relative to spinner or fermenter systems: relatively low cost, simplicity of construction leading to very low frequency of contamination, and, compared to fermenters, the ability to perform successive infections without interruption for cleaning and sterilization of equipment. Roller bottles may offer similar advantages [41,45,46].…”

Section: Discussionmentioning

confidence: 99%

Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domain

Madden¹,

Abele²,

Andersson³

et al. 2000

Eur J Biochem

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The ionotropic glutamate receptors (GluR) are the primary mediators of excitatory synaptic transmission in the brain. GluR agonist binding has been localized to an extracellular domain whose core is homologous to the bacterial periplasmic binding proteins (PBP). We have established routine, baculovirus-mediated expression of a complete ligand-binding domain construct at the 10-L scale, yielding 10±40 milligrams of purified protein. This construct contains peptides that lie outside the PBP-homologous core and that connect the domain core to the transmembrane domains of the channel and to the N-terminal`X'-domain. These linker peptides have been implicated in modulating channel physiology. Such extended constructs have proven difficult to express in bacteria, but the protein described here is stable and monomeric. Isothermal titration calorimetry reveals that glutamate binding to the domain involves a substantial heat capacity change and that at physiological temperatures, the reaction is both entropically and enthalpically favorable.Keywords: glutamate receptor ion channels; calorimetry; baculovirus/insect-cell expression; protein purification; ligand-binding thermodynamics.Glutamate receptor ion channels (GluR) are responsible for most excitatory synaptic communication in the central nervous system. They play an important role in the regulation of synaptic strength and are involved in a number of neuropathological processes, including post-traumatic neuronal damage, epilepsy and neurodegenerative diseases. GluR can be divided into three subclasses based on their pharmacological selectivity for the agonists a-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA), kainate and N-methyl-d-aspartate (NMDA) (reviewed in [1]). Like many ion channels, the GluR exhibit desensitization [2], the kinetics and extent of which depend on the agonist used.The GluR ligand-binding domain (`S1S2') is formed by two extracellular peptide sequences, known as S1 and S2, each approximately 150 amino acids long [3±5]. S1 is located N-terminal to the first transmembrane domain, and S2 is between the second and third transmembrane domains. The core of the ligand-binding domain, consisting of 80±85% of S1S2, exhibits homology to the prokaryotic periplasmic binding proteins (PBP). The PBP bind their ligands between two lobes, trapping them by a`Venus flytrap'-style cleft closure[6] (reviewed in [7]), although more restricted conformational changes are also observed, particularly among oligomeric members of the PBP family [8,9]. Sequences lacking PBP homology are located at both ends of the S1 and S2 peptides and connect the core of the domain to the`X'-domain located at the N-terminus and to the three transmembrane domains. These peptides have been implicated in modulating the electrophysiological characteristics of the channel and include the`flip/flop' alternative splice sequence important for desensitization kinetics [10±12].A fusion protein, in which the complete S1 and S2 sequences of the AMPA receptor GluRD are linked, reproduces the...

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Section: Discussionmentioning

confidence: 99%

Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domain

Madden¹,

Abele²,

Andersson³

et al. 2000

Eur J Biochem

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“…NK cells were used directly or cultured for 10 to 14 days with 50 units/mL human recombinant IL-2 (Sigma, St Louis, MO). Whole cell lysates were assayed for cathepsin C or granzyme B activity using the colorimetric substrates Gly-Phe-pNA (Sigma) and Ac-IEPD-pNA (Calbiochem, San Diego, CA), respectively, 14,15 or used in immunoblotting experiments. Aprotinin agarose binding was performed as described.…”

Section: Functional Studiesmentioning

confidence: 99%

A family with Papillon-Lefèvre syndrome reveals a requirement for cathepsin C in granzyme B activation and NK cell cytolytic activity

Meade

Wynter

Brett

et al. 2006

Blood

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IntroductionNatural killer (NK) cells are cytolytic lymphocytes whose function is to control infection and malignancy. Granule exocytosis delivers granzymes, a family of pro-apoptotic serine proteases, to target cells leading to the induction of caspasedependent and caspase-independent apoptosis. 1 Mouse granzyme B is required for the rapid induction of apoptosis in target cells. 2 However, other granzyme molecules contribute to the overall cytolytic phenotype, 3 with human cells expressing granzymes A, B, H, K, and M. 1,4 Activation of granzymes requires the removal of an N-terminal dipeptide. 5 Cathepsin C, a cysteine protease also known as dipeptidyl peptidase I (DPPI), has been implicated in granzyme A and B processing. 6,7 Cathepsin C-deficient mice fail to process granzymes A and B and hence lack both NK and T-cell-mediated cytolytic activity. 7 Human cathepsin C deficiency is the cause of Papillon-Lefèvre syndrome (PLS). [8][9][10] This is an extremely rare, autosomal recessive condition characterized by palmoplantar keratosis and severe periodontitis. In addition, skin infections and liver abscesses have been reported in PLS, 10,11 strongly suggesting an immunodeficiency component to the disease. However, in contrast to the mouse model, interleukin-2 (IL-2)-generated lymphokine activated killer (LAK) cell activity in these patients appears to be normal, and granzyme B is active in PLS LAK cells. 12 Here, we have tested the requirement for cathepsin C in the processing of granzyme B in unstimulated human NK cells from PLS patients. We describe an NK cell cytolytic defect and a failure of granzyme B activation. Study design PatientsPLS patients 1 and 2 are brother and sister from a consanguineous family, family 6 in Toomes et al. 8 Both patients are homozygous for a mutation changing glycine 277 of cathepsin C to a serine (G277S). This nomenclature is that used in the structural study 13 and refers to cathepsin C with the 24 amino-acid signal peptide removed. A third family member is unaffected by PLS and has one G277S allele and one wild-type allele and is referred to as the heterozygote. Blood samples from patients, the heterozygote, and healthy control donors were collected with informed consent and approval from the Leeds Teaching Hospital NHS Trust UK ethics committee. Functional studiesNK cells were phenotyped in whole blood using flow cytometry and isolated from peripheral blood mononuclear cells (PBMCs) using the NK isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). NK cells were used directly or cultured for 10 to 14 days with 50 units/mL human recombinant IL-2 (Sigma, St Louis, MO). Whole cell lysates were assayed for cathepsin C or granzyme B activity using the colorimetric substrates Gly-Phe-pNA (Sigma) and Ac-IEPD-pNA (Calbiochem, San Diego, CA), respectively, 14,15 or used in immunoblotting experiments. Aprotinin agarose binding was performed as described. 16,17 Killing and caspase activation assays were performed using flow cytometry, using CellTracker dye (Molecular Probes, Eug...

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“…Aliquots were withdrawn from the preincubation mixture at the indicated time points, and the activity was monitored in assay buffer containing 20 mM citric acid, pH 4.5, 150 mM NaCl, 1 mM EDTA, and 5 mM dithiothreitol with 0.1% Gly-Phe-pNA. are unknown, but it has been shown that proCatC can be efficiently activated with papain, and cathepsin L and S (Lauritzen et al, 1998;Dahl et al, 2001). Therefore, proCatC was treated with papain to mimic the in vivo proteolytic process and activate the proenzyme into its mature form, which led to active recombinant human CatC being successfully obtained.…”

Section: Discussionmentioning

confidence: 99%

“…It was previously demonstrated that maturation of proCatC with papain results in a 2000-fold increase in activity (Lauritzen et al, 1998). On the basis of this observation, proCatC was treated with papain to mimic the in vivo proteolytic process and to exert its proteinase activity.…”

Section: Expression and Activation Of Recombinant Human Catcmentioning

confidence: 99%

“…Therefore, production of recombinant human CatC protein in an effort to develop inhibitors of this enzyme is highly appreciated. In addition to this importance, due to its unique proteolytic specificity in sequential removal of dipeptides from the amino terminus of protein substrates, attention has also been drawn to the industrial applications of CatC as a processing exopeptidase to use in amino acid sequencing of proteins and removal of fusion peptides from the N-termini of recombinant proteins (Lauritzen et al, 1998;Pedersen et al, 1999).…”

Section: Introductionmentioning

confidence: 99%

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Cloning, expression, and activity analysis of human cathepsin C in the yeast Pichia pastoris

Daglioglu¹

2017

Turk J Biol

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The yeast Pichia pastoris expression system was investigated for the production of human cathepsin C (CatC) recombinant protein. The full-length CatC cDNA, corresponding to amino acids 12-475, was synthesized from interleukin-2 (IL-2) stimulated human peripheral blood mononuclear cells and subcloned in the pGEM-T cloning vector. After confirming the DNA sequence of the insert, the gene was cloned into the pPICZαA expression vector under the control of the methanol-inducible alcohol oxidase (AOX1) promoter and transformed to P. pastoris X-33 cells. The expressed protein was secreted into the culture medium through the α-factor mating signal sequence of the expression vector. Analysis of the culture supernatant revealed that the recombinant human CatC was secreted as a 58-kDa molecule, indicating that human CatC was accumulated in the culture supernatant as proform composed of the residual propart, the activation peptide, and the heavy and light chains. Extracellular recombinant proCatC was further activated by cysteine endoprotease papain in vitro and its activity was confirmed by assays using a synthetic substrate.

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Active Recombinant Rat Dipeptidyl Aminopeptidase I (Cathepsin C) Produced Using the Baculovirus Expression System

Cited by 32 publications

References 30 publications

Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domain

Large-scale expression and thermodynamic characterization of a glutamate receptor agonist-binding domain

A family with Papillon-Lefèvre syndrome reveals a requirement for cathepsin C in granzyme B activation and NK cell cytolytic activity

Cloning, expression, and activity analysis of human cathepsin C in the yeast Pichia pastoris

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