Purification and characterization of recombinant human cathepsin E expressed in human kidney cell line 293

Cappiello, Maria Grazia; Wu, Zhenyong; Scott, Boyd B.; McGeehan, Gerard M.; Harrison, Richard K.

doi:10.1016/j.pep.2004.05.013

Cited by 6 publications

(3 citation statements)

References 18 publications

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“…Using immunofluorescence, procathepsin E was detected throughout the cytoplasm and colocalized with early endosomal antigen 1 (EEA1) (Figure 1C). Late endosomes have a pH <6.0 and auto-activation of procathepsin E to the mature form occurs between pH 3.0 and pH 6.0 (Rao-Naik et al, 1995; Cappiello et al, 2004). Therefore it is unlikely that procathepsin E proceeds into late endosomes as the mature enzyme is not detected in the whole cell lysate.…”

Section: Resultsmentioning

confidence: 99%

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors

O’Donoghue

Ivry

Chaudhury

et al. 2016

Biological Chemistry

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The cathepsin family of lysosomal proteases is increasingly being recognized for their altered expression in cancer and role in facilitating tumor progression. The aspartyl protease cathepsin E is overexpressed in several cancers and has been investigated as a biomarker for pancreatic ductal adenocarcinoma (PDAC). Here we show that cathepsin E expression in mouse PDAC tumors is increased by more than 400-fold when compared to healthy pancreatic tissue. Cathepsin E accumulates over the course of disease progression and accounts for more than 3% of the tumor protein in mice with end-stage disease. Through immunoblot analysis we determined that only procathepsin E exists in mouse PDAC tumors and cell lines derived from these tumors. By decreasing the pH, this procathepsion E is converted to the mature form, resulting in an increase in proteolytic activity. Although active site inhibitors can bind procathepsin E, treatment of PDAC mice with the aspartyl protease inhibitor ritonavir did not decrease tumor burden. Lastly, we used multiplex substrate profiling by mass spectrometry to identify two synthetic peptides that are hydrolyzed by procathepsin E near neutral pH. This work represents a comprehensive analysis of procathepsin E in PDAC and could facilitate the development of improved biomarkers for disease detection.

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Section: Resultsmentioning

confidence: 99%

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors

O’Donoghue

Ivry

Chaudhury

et al. 2016

Biological Chemistry

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“…In order to increase the secretion levels of rHuOVGP1, HEK293 cells were used instead. In the past, HEK293 cells have been successfully used for the production of several recombinant human glycoproteins such as cathepsin E (Cappiello et al 2004) and IFNα2b (Loignon et al 2008). The use of HEK293 cells is also compatible with the large-scale cell cultivating system CELLSPIN as these cells can be grown in suspension.…”

Section: Discussionmentioning

confidence: 99%

Recombinant human oviductin regulates protein tyrosine phosphorylation and acrosome reaction

Zhao¹,

Yang²,

Jia³

et al. 2016

Reproduction

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The mammalian oviduct synthesizes and secretes a major glycoprotein known as oviductin (OVGP1), which has been shown to interact with gametes and early embryos. Here we report the use of recombinant DNA technology to produce, for the first time, the secretory form of human OVGP1 in HEK293 cells. HEK293 colonies stably expressing recombinant human OVGP1 (rHuOVGP1) were established by transfecting cells with an expression vector pCMV6-Entry constructed with OVGP1 cDNA. Large quantities of rHuOVGP1 were obtained from the stably transfected cells using the CELLSPIN cell cultivation system. A two-step purification system was carried out to yield rHuOVGP1 with a purity of >95%. Upon gel electrophoresis, purified rHuOVGP1 showed a single band corresponding to the 120-150 kDa size range of human OVGP1. Mass spectrometric analysis of the purified rHuOVGP1 revealed its identity as human oviductin. Immunofluorescence showed the binding of rHuOVGP1 to different regions of human sperm cell surfaces in various degrees of intensity. Prior treatment of sperm with 1% Triton X-100 altered the immunostaining pattern of rHuOVGP1 with an intense immunostaining over the equatorial segment and post-acrosomal region as well as along the length of the tail. Addition of rHuOVGP1 in the capacitating medium further enhanced tyrosine phosphorylation of sperm proteins in a time-dependent manner. After 4-h incubation in the presence of rHuOVGP1, the number of acrosome-reacted sperm induced by calcium ionophore significantly increased. The successful production of rHuOVGP1 can now facilitate the study of the role of human OVGP1 in fertilization and early embryo development.

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“…These inhibitors included commercially available statines and the small molecule inhibitor aliskiren. Pepstatin A (Isovaleryl-Val-Val-Sta-Ala-Sta) is a potent inhibitor of several aspartic proteases including cathepsin E (IC 50 of 0.2 nM [ 15 ]), cathepsin D (IC 50 of 0.4 nM [ 16 ]) and HIV protease (IC 50 of 250 μM [ 17 ]). Human renin is poorly inhibited by pepstatin A (IC 50 of 15 μM [ 18 ] and 36 μM [ 19 ]), while porcine renin exhibits sub-micromolar inhibition constants with this compound (IC 50 of 0.32 μM [ 18 ] and 0.58 μM [ 20 ]).…”

Section: Resultsmentioning

confidence: 99%

Purification and characterization of recombinant human renin for X-ray crystallization studies

Wu¹,

Cappiello²,

Scott³

et al. 2008

BMC Biochemistry

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Background: The renin-angiotensin-aldosterone system (RAS) cascade is a major target for the clinical management of hypertension. Although inhibitors of various components of this cascade have been developed successfully, development of renin inhibitors has proven to be problematic. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. X-ray crystallographic data could greatly assist the design and development of these inhibitors. Here we describe the purification and characterization of recombinant human renin for x-ray crystallization studies.

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Purification and characterization of recombinant human cathepsin E expressed in human kidney cell line 293

Cited by 6 publications

References 18 publications

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors

Procathepsin E is highly abundant but minimally active in pancreatic ductal adenocarcinoma tumors

Recombinant human oviductin regulates protein tyrosine phosphorylation and acrosome reaction

Purification and characterization of recombinant human renin for X-ray crystallization studies

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