Eukaryotic expression and secretion of EGFP-labeled annexin A5

Stöcker, Michael; Pardo, Alessa; Hetzel, Christian; Reutelingsperger, Chris; Fischer, Rainer; Barth, Stefan

doi:10.1016/j.pep.2007.12.009

Cited by 20 publications

(27 citation statements)

References 22 publications

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“…In the presence of calcium, annexin V (anxV) specifically binds to PS with high affinity, which can be used to detect PS externalization occurring on the membrane of apoptotic cells (Stocker et al 2008;van Engeland et al 1998). Thus, anxV can be used as an apoptosis marker, being applied in conjunction with propidium iodide (PI) to distinguish early apoptotic cells from viable cells as well as late apoptotic and necrotic cells through flow cytometry (FCM) (Peirouvi et al 2007;Sgonc and Gruber 1998;Stocker et al 2008).…”

Section: Introductionmentioning

confidence: 99%

“…Thus, anxV can be used as an apoptosis marker, being applied in conjunction with propidium iodide (PI) to distinguish early apoptotic cells from viable cells as well as late apoptotic and necrotic cells through flow cytometry (FCM) (Peirouvi et al 2007;Sgonc and Gruber 1998;Stocker et al 2008).…”

Section: Introductionmentioning

confidence: 99%

“…Preparation of fluorescein-labeled anxV, such as anxV-fluorescein isothiocyanate (FITC), involves protein expression and purification, chemical coupling reactions, and further exclusion of free fluorescein, requiring multiple manipulations and precise control (Brumatti et al 2008;Ernst et al 1998;Kuypers et al 1996;Stocker et al 2008;Tait et al 2006). Moreover, the amine-directed chemical modification of anxV will reduce its membrane-binding activity (Tait et al 2006).…”

mentioning

confidence: 99%

“…Moreover, the amine-directed chemical modification of anxV will reduce its membrane-binding activity (Tait et al 2006). The final products are heterogeneous mixtures of differentially labeled proteins, while fusion proteins such as anxVenhanced green fluorescent protein (EGFP) are brighter, more homogeneous, and photostable (Ernst et al 1998;Kurschus et al 2009;Stocker et al 2008;Tait et al 2006). Nowadays, at least 18 companies can provide EGFPlabeled anxV for applications.…”

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confidence: 99%

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Quantitative analysis of annexin V–membrane interaction by flow cytometry

Wang

Chen

et al. 2015

Eur Biophys J

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We constructed a green fluorescent phosphatidylserine (PS)-binding probe, which was generated by fusing enhanced green fluorescent protein (EGFP) to the C terminus of human annexin V (anxV). With this probe, we investigated anxV-membrane interaction under different calcium and anxV-EGFP concentrations through flow cytometry (FCM). A mathematical description of the binding characteristics is proposed and validated to quantify the relationship concerning the relative concentration of membrane-bound anxV (B), calcium concentration ([C]), and protein concentration ([P]). Further analyses reveal that [Formula: see text] is linear with [Formula: see text] or [Formula: see text] when [P] and [C] are fixed, respectively, which indicates that the anxV-membrane binding reaction may involve sequential multiple steps. Our study provides a reference for application of anxV in apoptosis detection. The mathematical expression facilitates exploration of the possible interactions between calcium, anxV, and membrane. The corresponding mathematical analysis strengthens the interpretation of the interaction data.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Quantitative analysis of annexin V–membrane interaction by flow cytometry

Wang

Chen

et al. 2015

Eur Biophys J

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“…The PEDF sequence was amplified from the cDNA of APRE-19 cells with gene-specific primers and extended by appropriate restriction enzyme sequences for subsequent cloning into pMS plasmids as described previously. 44,45 In the first PCR reaction the PEDF gene was fitted with NheI (n-primer-nhe-pedf: 5 0 -acccaagctg gctagccaccATGCAGGCCCTGGTGCTACTC-3 0 ) and NotI (c-primer-pedf-not: 5 0 -gttcgggccctgcggccgcGGGGCCCC TGGGGTCCAGAATC-3 0 ) restriction sites. This enabled the insertion of the PEDF gene with its native leader sequence by NheI/NotI restriction-mediated cloning into the plasmids pMS-L-A5-EGFP-MH and pMS-L-EGFP-A5-H-IV, 44 resulting in the two plasmids pMS-N-PEDF-EGFP-MH, which encoded a C-terminal PEDF fused to EGFP followed by a tandem Myc/His-tag, and pMS-N-PEDF-H-IV, which encoded PEDF only fitted with a His-…”

Section: Two Plasmids Expressing Pedf Proteins Fused To Egfp (Pms-n-pmentioning

confidence: 99%

High efficiency non-viral transfection of retinal and iris pigment epithelial cells with pigment epithelium-derived factor

et al. 2009

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Transplantation of pigment epithelial cells in patients with age-related macular degeneration and Parkinson's disease has the potential to improve functional rehabilitation. Genetic modification of cells before transplantation may allow the delivery of neuroprotective factors to achieve functional improvement. As transplantation of cells modified using viral vectors is complicated by the possible dissemination of viral particles and severe immune reactions, we have explored non-viral methods to insert genetic material in pigment epithelial cells. Using lipofection or nucleofection ARPE-19 cells, freshly isolated and primary retinal and iris pigment epithelial (IPE) cells were transfected with plasmids encoding green fluorescent protein (GFP) and with three plasmids encoding recombinant pigment epithelium-derived factor (PEDF) and GFP. Transfection efficiency was evaluated by fluorescence microscopy and stability of protein expression by immunoblotting. Pigment epithelial cells were successfully transfected with plasmid encoding GFP. Expression of GFP in ARPE-19 was transient, but was observed for up to 1 year in IPE cells. Analysis of pigment epithelial cells transfected with PEDF plasmids revealed that PEDF fusion proteins were successfully expressed and functionally active. In conclusion, efficient transfer of genetic information in pigment epithelial cells can be achieved using non-viral transfection protocols

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A novel approach for targeted elimination of CSPG4‐positive triple‐negative breast cancer cells using a MAP tau‐based fusion protein

Amoury

Mladenov

Nachreiner

et al. 2016

Intl Journal of Cancer

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Chondroitin sulfate proteoglycan 4 (CSPG4) has been identified as a highly promising target antigen for immunotherapy of triple-negative breast cancer (TNBC). TNBC represents a highly aggressive heterogeneous group of tumors lacking expression of estrogen, progesterone and human epidermal growth factor receptor 2. TNBC is particularly prevalent among young premenopausal women. No suitable targeted therapies are currently available and therefore, novel agents for the targeted elimination of TNBC are urgently needed. Here, we present a novel cytolytic fusion protein (CFP), designated aCSPG4(scFv)-MAP, that consists of a high affinity CSPG4-specific single-chain antibody fragment (scFv) genetically fused to a functionally enhanced form of the human microtubule-associated protein (MAP) tau. Our data indicate that aCSPG4(scFv)-MAP efficiently targets CSPG41 TNBC-derived cell lines MDA-MB-231 and Hs 578T and potently inhibits their growth with IC 50 values of 200 nM. Treatment with aCSPG(scFv)-MAP resulted in induction of the mitochondrial stress pathway by activation of caspase-9 as well as endonuclease G translocation to the nucleus, while induction of the caspase-3 apoptosis pathway was not detectable. Importantly, in vivo studies in mice bearing human breast cancer xenografts revealed efficient targeting to and accumulation of aCSPG4(scFv)-MAP at tumor sites resulting in prominent tumor regression. Taken together, this preclinical proof of concept study confirms the potential clinical value of aCSPG4(scFv)-MAP as a novel targeted approach for the elimination of CSPG4-positive TNBC.

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Eukaryotic expression and secretion of EGFP-labeled annexin A5

Cited by 20 publications

References 22 publications

Quantitative analysis of annexin V–membrane interaction by flow cytometry

Quantitative analysis of annexin V–membrane interaction by flow cytometry

High efficiency non-viral transfection of retinal and iris pigment epithelial cells with pigment epithelium-derived factor

A novel approach for targeted elimination of CSPG4‐positive triple‐negative breast cancer cells using a MAP tau‐based fusion protein

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