scholarly journals Single-step purification of full-length human androgen receptor

2005 ◽  
Vol 3 (1) ◽  
pp. nrs.03001 ◽  
Author(s):  
Dalia Juzumiene ◽  
Ching-yi Chang ◽  
Daju Fan ◽  
Tanya Hartney ◽  
John D. Norris ◽  
...  
Keyword(s):  
Androgen Receptor ◽  
Affinity Chromatography ◽  
Spodoptera Frugiperda ◽  
Single Step ◽  
Full Length ◽  
Structural Studies ◽  
Site Specific ◽  
E Coli ◽  
Human Androgen Receptor

The full-length human androgen receptor with an N-terminal biotin acceptor peptide tag was overexpressed in Spodoptera frugiperda cells in the presence of 1 μM dihydrotestosterone. Site-specific biotinylation of BAP was achieved in vivo by co-expression of E. coli biotin holoenzyme synthetase. The androgen receptor was purified by single-step affinity chromatography using Streptavidin Mutein Matrix under native conditions. The resultant protein was active, stable, 95% homogeneous, and we obtained sufficient yield for use in functional and structural studies.

scholarly journals CUDC-101, a Novel Inhibitor of Full-Length Androgen Receptor (flAR) and Androgen Receptor Variant 7 (AR-V7) Activity: Mechanism of Action and In Vivo Efficacy

2016 ◽  
Vol 7 (3) ◽  
pp. 196-210 ◽  
Author(s):  
Huiying Sun ◽  
Sanjay N. Mediwala ◽  
Adam T. Szafran ◽  
Michael A. Mancini ◽  
Marco Marcelli
Keyword(s):  
Androgen Receptor ◽  
Mechanism Of Action ◽  
Full Length ◽  
In Vivo Efficacy ◽  
Activity Mechanism

Biologic consequences of androgen receptor (AR) activity in MDV3100-resistant LNCaP cells and dependence on AR full length.

2014 ◽  
Vol 32 (4_suppl) ◽  
pp. 74-74
Author(s):  
Yoshiaki Yamamoto ◽  
Yohann Loriot ◽  
Eliana Beraldi ◽  
Tianyuan Zhou ◽  
Youngsoo Kim ◽  
...  
Keyword(s):  
Gene Expression ◽  
Androgen Receptor ◽  
Cell Growth ◽  
Functional Role ◽  
Full Length ◽  
Lncap Cells ◽  
Exon 1

74 Background: While recent reports link androgen receptor (AR) variants (AR-Vs) to castration resistant prostate cancer (CRPC), the biological significance of AR-Vs in AR-regulated cell survival and proliferation, independent of AR full length (AR-FL), remains controversial. To define the functional role of AR-FL and AR-Vs in MDV3100-resistant (MDV-R), we designed antisense oligonucleotide (ASO) targeting exon 1 and exon 8 in AR to knockdown AR-FL alone or in combination with AR-Vs and examined these effects in MDV-R LNCaP-derived cells in vitro and in vivo. Methods: We generated by selection MDV-R LNCaP-derived sub-lines that uniformly expressed high levels of both AR-FL and AR-V7 compared to CRPC LNCaP xenografts. Cell growth rates, protein and gene expression were analyzed using crystal violet assay, western blotting and real-time PCR, respectively. Exon 1 and 8 AR-ASO were evaluated in MDV-R49F CRPC LNCaP xenografts. Results: AR-V7 was transiently transfected in MDV-R49F cells and differential knockdown of AR-V7 and/or AR-FL by exon 1 versus exon 8 AR-ASO was used to evaluate relative biologic contributions of AR-FL versus AR-V7 in MDV-R LNCaP AR-V7 overexpressing cells. Exon 1 and 8 AR-ASO treatment in these cells similarly decreased prostate-specific antigen (PSA) expression and induced apoptosis as measured by caspase-3 and PARP cleavage and cell growth inhibition. To further define the functional role of AR-Vs in MDV-R LNCaP cells, we used a CE3 siRNA that specifically silenced AR-V7, but not AR-FL in MDV-R LNCaP cells. AR-V7 knockdown did not decrease PSA levels, did not induce apoptosis, and did not inhibit cell growth. In MDV-R LNCaP cells, exon 1 and 8 ASO similarly suppressed cell growth and AR-regulated gene expression in vitro and in vivo. Conclusions: These results indicate that the AR remains an important driver of MDV3100 resistance and, the biologic consequences mainly driven by AR-FL in MDV-R LNCaP models.

scholarly journals Optimized sequential purification protocol for in vivo site-specific biotinylated full-length dengue virus capsid protein

2013 ◽  
Vol 26 (5) ◽  
pp. 377-387
Author(s):  
M. K. Chong ◽  
K. Parthasarathy ◽  
H. Y. Yeo ◽  
M. L. Ng
Keyword(s):  
Dengue Virus ◽  
Capsid Protein ◽  
Full Length ◽  
Virus Capsid ◽  
Site Specific ◽  
Virus Capsid Protein ◽  
Purification Protocol

scholarly journals Testosterone modulates mitochondrial aconitase in the full-length human androgen receptor-transfected PC-3 prostatic carcinoma cells

2004 ◽  
Vol 33 (1) ◽  
pp. 121-132 ◽  
Author(s):  
HH Juang ◽  
ML Hsieh ◽  
KH Tsui
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Androgen Receptor ◽  
Enzymatic Activity ◽  
Prostatic Carcinoma ◽  
Transient Gene Expression ◽  
Carcinoma Cells ◽  
Full Length ◽  
Mitochondrial Aconitase ◽  
Human Androgen Receptor

In vitro studies indicated that dihydrotestosterone (DHT) stimulates the enzymatic activity of the mitochondrial aconitase (mACON) in androgen-sensitive prostatic carcinoma cells, LNCaP. Cell proliferation assay determined that DHT doubles the optimal proliferation response of LNCaP cells. The androgen-insensitive human prostatic carcinoma cells, PC-3, were overexpressed in the human androgen receptor to assess the involvement of the native androgen receptor in the regulation by DHT of mACON gene expression. A stable-transfected clone that expresses the full-length androgen receptor was selected and termed PCAR9. The results revealed that DHT-treated PCAR9 cells paradoxically not only reduced the enzymatic activity of mACON but also blocked the biosynthesis of intracellular ATP attenuating cell proliferation. Transient gene expression assay indicated that DHT divergently regulates the promoter activity of the mACON gene in LNCaP and PCAR9 cells. This study suggested that DHT regulates mACON gene expression and the proliferation of cells in a receptor-dependent model through modulation by unidentified non-receptor factors.

Androgen-dependent cell cycle arrest and apoptotic death in PC-3 prostatic cell cultures expressing a full-length human androgen receptor

1997 ◽  
Vol 126 (1) ◽  
pp. 59-73 ◽  
Author(s):  
Lawrence E Heisler ◽  
Andreas Evangelou ◽  
April M Lew ◽  
John Trachtenberg ◽  
Harry P Elsholtz ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Androgen Receptor ◽  
Cell Cycle Arrest ◽  
Cell Cultures ◽  
Full Length ◽  
Cycle Arrest ◽  
Apoptotic Death ◽  
Dependent Cell ◽  
Human Androgen Receptor ◽  
Prostatic Cell

scholarly journals The Escherichia coli Fis Protein Stimulates Bacteriophage λ Integrative Recombination In Vitro

2003 ◽  
Vol 185 (10) ◽  
pp. 3076-3080 ◽  
Author(s):  
Dominic Esposito ◽  
Gary F. Gerard
Keyword(s):  
Escherichia Coli ◽  
Host Cell ◽  
Essential Role ◽  
Bacteriophage Λ ◽  
Site Specific ◽  
Site Specific Recombination ◽  
E Coli ◽  
In Vivo Recombination

ABSTRACT The Escherichia coli nucleoid-associated protein Fis was previously shown to be involved in bacteriophage lambda site-specific recombination in vivo, enhancing the levels of both integrative recombination and excisive recombination. While purified Fis protein was shown to stimulate in vitro excision, Fis appeared to have no effect on in vitro integration reactions even though a 15-fold drop in lysogenization frequency had previously been observed in fis mutants. We demonstrate here that E. coli Fis protein does stimulate integrative lambda recombination in vitro but only under specific conditions which likely mimic natural in vivo recombination more closely than the standard conditions used in vitro. In the presence of suboptimal concentrations of Int protein, Fis stimulates the rate of integrative recombination significantly. In addition, Fis enhances the recombination of substrates with nonstandard topologies which may be more relevant to the process of in vivo phage lambda recombination. These data support the hypothesis that Fis may play an essential role in lambda recombination in the host cell.

scholarly journals Technical Data of Heterologous Expression and Purification of SARS-CoV-2 Proteases Using Escherichia coli System

Data ◽  
2021 ◽  
Vol 6 (9) ◽  
pp. 99
Author(s):  
Rafida Razali ◽  
Vijay Kumar Subbiah ◽  
Cahyo Budiman
Keyword(s):  
Affinity Chromatography ◽  
Drug Targets ◽  
Single Step ◽  
Host Cells ◽  
Soluble Form ◽  
Expression And Purification ◽  
Technical Data ◽  
E Coli ◽  
Main Protease ◽  
Chromatography Step

The SARS-CoV-2 coronavirus expresses two essential proteases: firstly, the 3Chymotrypsin-like protease (3CLpro) or main protease (Mpro), and secondly, the papain-like protease (PLpro), both of which are considered as viable drug targets for the inhibition of viral replication. In order to perform drug discovery assays for SARS-CoV-2, it is imperative that efficient methods are established for the production and purification of 3CLpro and PLpro of SARS-CoV-2, designated as 3CLpro-CoV2 and PLpro-CoV2, respectively. This article expands the data collected in the attempts to express SARS-CoV-2 proteases under different conditions and purify them under single-step chromatography. Data showed that the use of E. coli BL21(DE3) strain was sufficient to express 3CLpro-CoV2 in a fully soluble form. Nevertheless, the single affinity chromatography step was only applicable for 3CLpro-CoV2 expressed at 18 °C, with a yield and purification fold of 92% and 49, respectively. Meanwhile, PLpro-CoV2 was successfully expressed in a fully soluble form in either BL21(DE3) or BL21-CodonPlus(DE3) strains. In contrast, the single affinity chromatography step was only applicable for PLpro-CoV2 expressed using E. coli BL21-CodonPlus(DE3) at 18 or 37 °C, with a yield and purification fold of 86% (18 °C) or 83.36% (37 °C) and 112 (18 °C) or 71 (37 °C), respectively. The findings provide a guide for optimizing the production of SARS-CoV-2 proteases of E. coli host cells.

scholarly journals Synthesis of site-specific antibody-drug conjugates by ADP-ribosyl cyclases

2020 ◽  
Vol 6 (23) ◽  
pp. eaba6752 ◽  
Author(s):  
Zhefu Dai ◽  
Xiao-Nan Zhang ◽  
Fariborz Nasertorabi ◽  
Qinqin Cheng ◽  
Jiawei Li ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Reaction Times ◽  
Single Step ◽  
Her2 Positive ◽  
Site Specific ◽  
Antibody Drug Conjugates ◽  
Drug Conjugates ◽  
Antibody Drug

Most of the current antibody-drug conjugates (ADCs) in clinic are heterogeneous mixtures. To produce homogeneous ADCs, established procedures often require multiple steps or long reaction times. The introduced mutations or foreign sequences may cause high immunogenicity. Here, we explore a new concept of transforming CD38 enzymatic activity into a facile approach for generating site-specific ADCs. This was achieved through coupling bifunctional antibody-CD38 fusion proteins with designer dinucleotide-based covalent inhibitors with stably attached payloads. The resulting adenosine diphosphate–ribosyl cyclase–enabled ADC (ARC-ADC) with a drug-to-antibody ratio of 2 could be rapidly generated through single-step conjugation. The generated ARC-ADC targeting human epidermal growth factor receptor 2 (HER2) displays excellent stability and potency against HER2-positive breast cancer both in vitro and in vivo. This proof-of-concept study demonstrates a new strategy for production of site-specific ADCs. It may provide a general approach for the development of a novel class of ADCs with potentially enhanced properties.

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