Identification of a domain within MDMX-S that is responsible for its high affinity interaction with p53 and high-level expression in mammalian cells

2003 ◽  
Vol 89 (3) ◽  
pp. 563-575 ◽  
Author(s):  
Ravikumar Rallapalli ◽  
Gordon Strachan ◽  
Rocky S. Tuan ◽  
David J. Hall
Keyword(s):  
Mammalian Cells ◽  
High Level Expression ◽  
High Affinity ◽  
High Level ◽  
Affinity Interaction ◽  
Expression In Mammalian Cells

High level expression of the Drosophila Toll receptor ectodomain and crystallization of its complex with the morphogen Spätzle

2013 ◽  
Vol 394 (8) ◽  
pp. 1091-1096 ◽  
Author(s):  
Marco Stelter ◽  
Uwe Fandrich ◽  
Kati Franzke ◽  
Angelika Schierhorn ◽  
Constanze Breithaupt ◽  
...  
Keyword(s):  
Immune Response ◽  
High Level Expression ◽  
Receptor Ligand ◽  
High Affinity ◽  
Cystine Knot ◽  
Toll Receptor ◽  
Cystine Knot Protein ◽  
High Yields ◽  
High Level ◽  
Affinity Interaction

Abstract Drosophila Toll receptors are involved in embryonic development and in the immune response of adult flies. In both processes, the Toll receptor ligand is the NGF-like cystine knot protein Spätzle. Here we present the expression of Toll receptor ectodomain in Schneider cells at high yields and demonstrate a high affinity interaction with the refolded and trypsin-processed Spätzle cystine knot domain dimer. Poorly and anisotropically diffracting crystals of the complex could be improved by deglycosylation and dehydration, paving the way for structural analyses of the Toll-Spätzle interaction.

High level expression, purification and activation of human dipeptidyl peptidase I from mammalian cells

2011 ◽  
Vol 76 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Wei Yang ◽  
Wenjuan Xia ◽  
Jingjing Mao ◽  
Daqi Xu ◽  
Jianhe Chen ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Dipeptidyl Peptidase ◽  
High Level Expression ◽  
High Level

scholarly journals Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC

2019 ◽  
Author(s):  
Binhui Zhao ◽  
Pankaj Chaturvedi ◽  
David L. Zimmerman ◽  
Andrew S. Belmont
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Large Fraction ◽  
Fine Tuning ◽  
Cmv Promoter ◽  
Chromosome Position ◽  
High Level ◽  
Genomic Regions ◽  
Expression In Mammalian Cells

ABSTRACTAchieving reproducible, stable, and high-level transgene expression in mammalian cells remains problematic. Previously, we attained copy-number-dependent, chromosome-position-independent expression of reporter minigenes by embedding them within a BAC containing the mouseMsh3-Dhfrlocus (DHFR BAC). Here we extend this “BAC TG-EMBED” approach. First, we report a toolkit of endogenous promoters capable of driving transgene expression over a 0.01-5 fold expression range relative to the CMV promoter, allowing fine-tuning of relative expression levels of multiple reporter genes expressed on a single BAC. Second, we show small variability in both the expression level and long-term expression stability of a reporter gene embedded in BACs containing either transcriptionally active or inactive genomic regions, making choice of BACs more flexible. Third, we describe an intriguing phenomenon in which BAC transgenes are maintained as episomes in a large fraction of stably selected clones. Finally, we demonstrate the utility of BAC TG-EMBED by simultaneously labeling three nuclear compartments in 94% of stable clones using a multi-reporter DHFR BAC, constructed with a combination of synthetic biology and BAC recombineering tools. Our extended BAC TG-EMBED method provides a versatile platform for achieving reproducible, stable simultaneous expression of multiple transgenes maintained either as episomes or stably integrated copies.

scholarly journals High-level expression and purification of a recombinant human erythropoietin produced using a baculovirus vector

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 652-657
Author(s):  
FW Quelle ◽  
LF Caslake ◽  
RE Burkert ◽  
DM Wojchowski
Keyword(s):  
Mammalian Cells ◽  
Recombinant Human Erythropoietin ◽  
Insect Cell Line ◽  
Recombinant Baculovirus ◽  
Cell Surface Receptor ◽  
High Level Expression ◽  
Human Erythropoietin ◽  
Highly Active ◽  
High Level

Conditions presently have been established for the high-level expression and simplified purification of recombinant human erythropoietin produced in Spodoptera frugiperda cells. Expression, as mediated by infection with a recombinant baculovirus, was accomplished in suspension culture using reduced levels of serum and media supplements experimentally determined to provide optimum levels of factor production (500,000 U/L). Purification of this recombinant human erythropoietin to virtual homogeneity (greater than or equal to 99%) was accomplished via a simple three-step procedure involving isocratic elution from DEAE-Sephacel, reverse-phase high performance liquid chromatography (HPLC) on a C4 medium, and the single-step elution of purified hormone from concanavalin A agarose. Overall, an 890-fold purification was accomplished with a recovery of 80% as assayed in vitro. Biologically, this purified erythropoietin is highly active, possessing a specific activity in vitro of 200,000 U/mg protein. Chemically, this erythropoietin (molecular weight [mol wt] 26,200) appears exceptionally uniform in its oligosaccharide constitution (30%) as contrasted with heterogeneously glycosylated erythropoietins derived from mammalian cells (mol wt 30,000 to 38,000; 40% to 50% complex-type oligosaccharide). Thus, human erythropoietin as presently produced in an insect cell line comprises not only an abundant source of highly active, readily purified hormone for studies of its mechanism of action and cell surface receptor, but also represents a uniquely homogeneous form that should prove advantageous for direct structural analyses.

[5] Stable high-level gene expression in mammalian cells by T7 phage RNA polymerase

1993 ◽  
pp. 47-66 ◽  
Author(s):  
Andre Lieber ◽  
Volker Sandig ◽  
Wolfgang Sommer ◽  
Silvia Bähring ◽  
Michael Strauss
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Mammalian Cells ◽  
High Level ◽  
T7 Phage ◽  
Expression In Mammalian Cells
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