Efficient and versatile one-step affinity purification of in vivo biotinylated proteins: Expression, characterization and structure analysis of recombinant human glutamate carboxypeptidase II

Through the combination of X-ray structure analysis and theoretical calculations at the B3LYP-D3/def2-TZVP level of theory, we designed and investigated two novel host units for the recognition of neutral (e.g....

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Expression of glutamate carboxypeptidase II in the glial tumor recurrence evaluated in vivo using radionuclide imaging

Scientific Reports ◽

10.1038/s41598-021-04613-w ◽

2022 ◽

Vol 12 (1) ◽

Author(s):

Jolanta Kunikowska ◽

Rafał Czepczyński ◽

Dariusz Pawlak ◽

Henryk Koziara ◽

Kacper Pełka ◽

...

Keyword(s):

Standard Uptake Value ◽

Recurrent Glioma ◽

Normal Brain ◽

Glial Tumor ◽

Glutamate Carboxypeptidase Ii ◽

Pet Ct ◽

Significant Difference ◽

Glutamate Carboxypeptidase

AbstractGlutamate carboxypeptidase II (GCP), also known as prostate specific membrane antigen (PSMA) has been found to be expressed in glioma vasculature in in-vitro studies. GCP expression can be traced with the use of [68Ga]Ga-PSMA-11 PET/CT used routinely for prostate cancer imaging. The aim of this paper was to analyze GCP expression in the recurrent glial tumors in vivo. 34 patients (pts.) aged 44.5 ± 10.3 years with suspicion of recurrence of histologically confirmed glioma grade III (6 pts.) and grade IV (28 pts.) were included in the study. All patients underwent contrast-enhanced MR and [68Ga]Ga-PSMA-11 PET/CT. No radiopharmaceutical-related adverse events were noted. PET/CT was positive in all the areas suspected for recurrence at MR in all the patients. The recurrence was confirmed by histopathological examinations or follow-up imaging in all cases. The images showed a very low background activity of the normal brain. Median maximal standard uptake value (SUVmax) of the tumors was 6.5 (range 0.9–15.6) and mean standard uptake value (SUVmean) was 3.5 (range 0.9–7.5). Target-to-background (TBR) ratios varied between 15 and 1400 with a median of 152. Target-to-liver background ratios (TLR) ranged from 0.2 to 2.6, the median TLR was 1.3. No significant difference of the measured parameters was found between the subgroups according to the glioma grade. High GCP expression in the recurrent glioma was demonstrated in-vivo with the use of [68Ga]Ga-PSMA-11 PET/CT. As the treatment options in recurrent glioma are limited, this observation may open new therapeutic perspectives with the use of radiolabeled agents targeting the GCP.

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In vivo Molecular Imaging of Glutamate Carboxypeptidase II Expression in Re-endothelialisation after Percutaneous Balloon Denudation in a Rat Model

Scientific Reports ◽

10.1038/s41598-018-25863-1 ◽

2018 ◽

Vol 8 (1) ◽

Cited By ~ 4

Author(s):

Heike Endepols ◽

Felix M. Mottaghy ◽

Sakine Simsekyilmaz ◽

Jan Bucerius ◽

Felix Vogt ◽

...

Keyword(s):

Molecular Imaging ◽

Rat Model ◽

Glutamate Carboxypeptidase Ii ◽

Percutaneous Balloon ◽

Glutamate Carboxypeptidase

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11C-MCG: Synthesis, Uptake Selectivity, and Primate PET of a Probe for Glutamate Carboxypeptidase II (NAALADase)

Molecular Imaging ◽

10.1162/15353500200202109 ◽

2002 ◽

Vol 1 (2) ◽

pp. 153535002002021

Author(s):

Martin G. Pomper ◽

John L. Musachio ◽

Jiazhong Zhang ◽

Ursula Scheffel ◽

Yun Zhou ◽

...

Keyword(s):

Radiochemical Yield ◽

Target Tissue ◽

Glutamatergic Transmission ◽

Glutamate Carboxypeptidase Ii ◽

Quantitative Pet ◽

Positron Emission ◽

Glutamate Carboxypeptidase ◽

Pentanedioic Acid ◽

The Brain

Imaging of glutamate carboxypeptidase II (GCP II), also known as N-acetylated α-linked l-amino dipeptidase (NAALADase), may enable study of glutamatergic transmission, prostate cancer, and tumor neovasculature in vivo. Our goal was to develop a probe for GCP II for use with positron emission tomography (PET). Radiosynthesis of 11C–MeCys–C(O)–Glu or 11C-( S)-2-[3-(( R)-1-carboxy-2-methylsulfanyl-ethyl)-ureido]-pentanedioic acid (11C-MCG), an asymmetric urea and potent ( Ki = 1.9 nM) inhibitor of GCP II, was performed by C-11 methylation of the free thiol. Biodistribution of 11C-MCG was assayed in mice, and quantitative PET was performed in a baboon. 11C-MCG was obtained in 16% radiochemical yield at the end of synthesis with specific radioactivities over 167 GBq/mmol (4000 Ci/mmol) within 30 min after the end of bombardment. At 30 min postinjection, 11C-MCG showed 33.0 ± 5.1%, 0.4 ± 0.1%, and 1.1 ± 0.2% ID/g in mouse kidney (target tissue), muscle, and blood, respectively. Little radioactivity gained access to the brain. Blockade with unlabeled MCG or 2-(phosphonomethyl)pentanedioic acid (PMPA), another potent inhibitor of GCP II, provided sevenfold and threefold reductions, respectively, in binding to target tissue. For PET, distribution volumes (DVs) were 1.38 then 0.87 pre- and postblocker (PMPA). Little metabolism of 11C-MCG occurred in the mouse or baboon. These results suggest that 11C-MCG may be useful for imaging GCP II in the periphery.

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Isolation of Human Fibrinogen and its Derivatives by Affinity Chromatography on Gly-Pro-Arg-Pro-Lys-Fractogel

Thrombosis and Haemostasis ◽

10.1055/s-0038-1645062 ◽

1990 ◽

Vol 63 (03) ◽

pp. 439-444 ◽

Cited By ~ 19

Author(s):

C Kuyas ◽

A Haeberli ◽

P Walder ◽

P W Straub

Keyword(s):

Gel Electrophoresis ◽

Affinity Purification ◽

Polyacrylamide Gel Electrophoresis ◽

Ease Of Use ◽

Terminal Sequence ◽

Purification Method ◽

Human Fibrinogen ◽

Isolation Methods ◽

One Step ◽

Complementary Binding

SummaryWith an immobilized synthetic pentapeptide GlyProArgProLys comprising the N-terminal sequence GlyProArg of the α-chain of fibrin, a new affinity method for the quantitative isolation of fibrinogen out of anticoagulated plasma was developed. The method proved to be superior to all known isolation methods in respect to ease of use and yield, since fibrinogen could be isolated in one step out of plasma with a recovery of more than 95% when compared to the immunologically measurable amounts of fibrinogen. Moreover the amounts of contaminating proteins such as fibronectin, factor XIII or plasminogen were negligible and the purity of the isolated fibrinogen was higher than 95% as measured by polyacrylamide gel electrophoresis. The clottability was 90% and more. Another advantage of this affinity purification method is the possibility to isolate fibrinogen quantitatively out of small plasma samples (<5 ml). Further, abnormal fibrinogen molecules, provided their complementary binding site for GlyProArg is preserved, may also be quantitatively isolated independent of any solubility differences as compared to normal fibrinogen. In addition fibrin(ogcn) fragments originating from plasmic digestion can be separated on the basis of their affinity to GlyProArg. The described affinity gel can be used more than 50 times without any loss of capacity.

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One-step Affinity Purification of the Chloroplast ClpP Complex from the Green Alga Chlamydomonas reinhardtii Using the Strep-tagII Epitope Tag

BIO-PROTOCOL ◽

10.21769/bioprotoc.315 ◽

2013 ◽

Vol 3 (1) ◽

Author(s):

Benoit Derrien ◽

Olivier Vallon

Keyword(s):

Chlamydomonas Reinhardtii ◽

Green Alga ◽

Affinity Purification ◽

Epitope Tag ◽

One Step

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USP9X-mediated KDM4C deubiquitination promotes lung cancer radioresistance by epigenetically inducing TGF-β2 transcription

Cell Death and Differentiation ◽

10.1038/s41418-021-00740-z ◽

2021 ◽

Author(s):

Xiaohua Jie ◽

William Pat Fong ◽

Rui Zhou ◽

Ye Zhao ◽

Yingchao Zhao ◽

...

Keyword(s):

Lung Cancer ◽

Affinity Purification ◽

Smad Signaling ◽

Lung Cancer Patients ◽

Lysine Specific Demethylase ◽

Underlying Mechanisms ◽

H3k9me3 Level ◽

Critical Binding

AbstractRadioresistance is regarded as the main barrier to effective radiotherapy in lung cancer. However, the underlying mechanisms of radioresistance remain elusive. Here, we show that lysine-specific demethylase 4C (KDM4C) is overexpressed and correlated with poor prognosis in lung cancer patients. We provide evidence that genetical or pharmacological inhibition of KDM4C impairs tumorigenesis and radioresistance in lung cancer in vitro and in vivo. Moreover, we uncover that KDM4C upregulates TGF-β2 expression by directly reducing H3K9me3 level at the TGF-β2 promoter and then activates Smad/ATM/Chk2 signaling to confer radioresistance in lung cancer. Using tandem affinity purification technology, we further identify deubiquitinase USP9X as a critical binding partner that deubiquitinates and stabilizes KDM4C. More importantly, depletion of USP9X impairs TGF-β2/Smad signaling and radioresistance by destabilizing KDM4C in lung cancer cells. Thus, our findings demonstrate that USP9X-mediated KDM4C deubiquitination activates TGF-β2/Smad signaling to promote radioresistance, suggesting that targeting KDM4C may be a promising radiosensitization strategy in the treatment of lung cancer.

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In Vivo Identification of Intermediate Stages of the DNA Inversion Reaction Catalyzed by the Salmonella Hin Recombinase

Genetics ◽

10.1093/genetics/149.4.1649 ◽

1998 ◽

Vol 149 (4) ◽

pp. 1649-1663

Author(s):

Oliver Z Nanassy ◽

Kelly T Hughes

Keyword(s):

Amino Acids ◽

Biochemical Analysis ◽

Mutational Analysis ◽

Recombination Reaction ◽

Recombination Site ◽

Dna Inversion ◽

Hin Recombinase ◽

One Step ◽

A Site

Abstract The Hin recombinase catalyzes a site-specific recombination reaction that results in the reversible inversion of a 1-kbp segment of the Salmonella chromosome. The DNA inversion reaction catalyzed by the Salmonella Hin recombinase is a dynamic process proceeding through many intermediate stages, requiring multiple DNA sites and the Fis accessory protein. Biochemical analysis of this reaction has identified intermediate steps in the inversion reaction but has not yet revealed the process by which transition from one step to another occurs. Because transition from one reaction step to another proceeds through interactions between specific amino acids, and between amino acids and DNA bases, it is possible to study these transitions through mutational analysis of the proteins involved. We isolated a large number of mutants in the Hin recombinase that failed to carry out the DNA exchange reaction. We generated genetic tools that allowed the assignment of these mutants to specific transition steps in the recombination reaction. This genetic analysis, combined with further biochemical analysis, allowed us to define contributions by specific amino acids to individual steps in the DNA inversion reaction. Evidence is also presented in support of a model that Fis protein enhances the binding of Hin to the hixR recombination site. These studies identified regions within the Hin recombinase involved in specific transition steps of the reaction and provided new insights into the molecular details of the reaction mechanism.

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