Characterization of three different single chain antibodies recognizing non-reducing terminal mannose residues expressed in Escherichia coli by an inducible T7 expression system

2011 ◽  
Vol 150 (4) ◽  
pp. 439-450 ◽  
Author(s):  
A. Matsumoto-Takasaki ◽  
N. Yuasa ◽  
D. Katagiri ◽  
T. Koyama ◽  
K. Sakai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Expression System ◽  
Single Chain ◽  
Single Chain Antibodies ◽  
T7 Expression System ◽  
Terminal Mannose

Isolation and Characterization of Phage-Displayed Single Chain Antibodies Recognizing Nonreducing Terminal Mannose Residues. 1. A New Strategy for Generation of Anti-Carbohydrate Antibodies†

Biochemistry ◽  
2007 ◽  
Vol 46 (1) ◽  
pp. 253-262 ◽  
Author(s):  
Keiko Sakai ◽  
Yoshitaka Shimizu ◽  
Tomoki Chiba ◽  
Ayano Matsumoto-Takasaki ◽  
Yu Kusada ◽  
...  
Keyword(s):  
Single Chain ◽  
Single Chain Antibodies ◽  
Isolation And Characterization ◽  
New Strategy ◽  
Terminal Mannose

Isolation and Characterization of Phage-Displayed Single Chain Antibodies Recognizing Nonreducing Terminal Mannose Residues. 2. Expression, Purification, and Characterization of Recombinant Single Chain Antibodies†

Biochemistry ◽  
2007 ◽  
Vol 46 (1) ◽  
pp. 263-270 ◽  
Author(s):  
Wei Zhang ◽  
Ayano Matsumoto-Takasaki ◽  
Yu Kusada ◽  
Hiroyuki Sakaue ◽  
Keiko Sakai ◽  
...  
Keyword(s):  
Single Chain ◽  
Purification And Characterization ◽  
Single Chain Antibodies ◽  
Isolation And Characterization ◽  
Terminal Mannose

scholarly journals Functional expression of the eukaryotic proton pump rhodopsin OmR2 in Escherichia coli and its photochemical characterization

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Masuzu Kikuchi ◽  
Keiichi Kojima ◽  
Shin Nakao ◽  
Susumu Yoshizawa ◽  
Shiho Kawanishi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Proton Pump ◽  
Expression System ◽  
Spectroscopic Characterization ◽  
Functional Expression ◽  
Proton Pumping ◽  
E Coli ◽  
Oxyrrhis Marina ◽  
Domains Of Life

AbstractMicrobial rhodopsins are photoswitchable seven-transmembrane proteins that are widely distributed in three domains of life, archaea, bacteria and eukarya. Rhodopsins allow the transport of protons outwardly across the membrane and are indispensable for light-energy conversion in microorganisms. Archaeal and bacterial proton pump rhodopsins have been characterized using an Escherichia coli expression system because that enables the rapid production of large amounts of recombinant proteins, whereas no success has been reported for eukaryotic rhodopsins. Here, we report a phylogenetically distinct eukaryotic rhodopsin from the dinoflagellate Oxyrrhis marina (O. marina rhodopsin-2, OmR2) that can be expressed in E. coli cells. E. coli cells harboring the OmR2 gene showed an outward proton-pumping activity, indicating its functional expression. Spectroscopic characterization of the purified OmR2 protein revealed several features as follows: (1) an absorption maximum at 533 nm with all-trans retinal chromophore, (2) the possession of the deprotonated counterion (pKa = 3.0) of the protonated Schiff base and (3) a rapid photocycle through several distinct photointermediates. Those features are similar to those of known eukaryotic proton pump rhodopsins. Our successful characterization of OmR2 expressed in E. coli cells could build a basis for understanding and utilizing eukaryotic rhodopsins.

Optimization of the expression of single-chain antibodies using different Escherichia coli systems

2007 ◽  
Vol 131 (2) ◽  
pp. S251-S252
Author(s):  
Symon Riedstra ◽  
Gonçalo Leite ◽  
Carolina Ferreira ◽  
Francisco Brás Gomes ◽  
Paulo M.P. Costa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Single Chain ◽  
Single Chain Antibodies

Construction and characterization of two anti-sweetener single chain antibodies using radioligand binding, fluorescence and circular dichroism spectroscopy

1999 ◽  
Vol 12 (4) ◽  
pp. 258-266 ◽  
Author(s):  
David W. Pledger ◽  
Thomas C. Brodnicki ◽  
Brent L. Graham ◽  
Sergey Tetin ◽  
David M. Kranz ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Radioligand Binding ◽  
Circular Dichroism Spectroscopy ◽  
Single Chain ◽  
Single Chain Antibodies ◽  
Circular Dichroïsm

Construction and characterization of mutated LEA peptides in Escherichia coli to develop an efficient protein expression system

2017 ◽  
Vol 31 (1) ◽  
pp. e2658 ◽  
Author(s):  
Nishit Pathak ◽  
Hiro Hamada ◽  
Shinya Ikeno
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Expression System ◽  
Protein Expression System

scholarly journals Development and Characterization of Nanobodies Targeting the Kupffer Cell

2021 ◽  
Vol 12 ◽  
Author(s):  
Fang Zheng ◽  
Jinhong Zhou ◽  
Zhenlin Ouyang ◽  
Jiaxin Zhang ◽  
Xinyi Wang ◽  
...  
Keyword(s):  
Kupffer Cell ◽  
Cell Receptor ◽  
Therapeutic Agents ◽  
Membrane Receptors ◽  
Single Chain ◽  
Single Chain Antibodies ◽  
Functional Investigation ◽  
And Function ◽  
Distinct Features

Nanobodies that are derived from single-chain antibodies of camelids have served as powerful tools in diagnostics, therapeutics and investigation of membrane receptors' structure and function. In this study, we developed a series of nanobodies by a phage display screening building from lymphocytes isolated from an alpaca immunized with recombinant mouse Kupffer cell receptor Clec4F, which is involved in pathogen recognition by binding to galactose and N-acetylgalactosamine. Bio-panning selections retrieved 14 different nanobodies against Clec4F with an affinity ranging from 0.2 to 2 nM as determined by SPR. Those nanobodies mainly recognize 4 different epitopes as analyzed via competitive epitope binning. By analysis of the radioactivity in each organ after injection of 99mTc labeled Clec4F nanobodies in naïve mice, we found that these nanobodies are targeting the liver. Furthermore, we performed a structural characterization at atomic resolution of two of the Clec4F nanobodies from different epitope groups, which revealed distinct features within the CDR2 and CDR3 regions. Taken together, we developed a series of nanobodies targeting multiple distinct recognition epitopes of the Kupffer cell-specific receptor Clec4F which may be useful for its structural and functional investigation as well as for use as molecular imaging and therapeutic agents.

scholarly journals High-level expression of soluble rat hsc70 in Escherichia coli: purification and characterization of the cloned enzyme

1993 ◽  
Vol 294 (1) ◽  
pp. 69-77 ◽  
Author(s):  
C Wang ◽  
M R Lee
Keyword(s):  
Escherichia Coli ◽  
Dissociation Constants ◽  
Expression System ◽  
Deae Cellulose ◽  
Basic Amino Acid ◽  
Coated Vesicle ◽  
Amino Acid Residues ◽  
High Level ◽  
Binding Component

We have cloned the cDNA of rat hsc70 (clathrin-uncoating ATPase) into a T7 expression system and have expressed this enzyme in Escherichia coli. The recombinant clathrin-uncoating ATPase is in the cytosolic fraction of the bacterium and is soluble. It was purified to homogeneity by DEAE-cellulose and ATP-agarose column chromatography. From 1 litre of bacterial culture (0.3-0.4 g of proteins), 5-20 mg of pure recombinant clathrin-uncoating ATPase was routinely obtained. The cloned enzyme is capable of dissociating clathrin from bovine coated vesicle. Furthermore, it is not methylated on basic amino acid residues and is not blocked at the N-terminus, indicating that these modifications on hsc70 are not essential for uncoating of clathrin. Binding of [alpha-32P]ATP by purified recombinant hsc70 was analysed by Scatchard plot. The results indicate that there one high-affinity binding component with a Kd (dissociation constant) of 0.2-0.3 microM. The peptide-stimulated ATPase activities of recombinant hsc70 at 37 degrees C with respect to S-peptide peptides P3a and GT4 at a concentration of 1.2 mM are 142 +/- 6, 214 +/- 8 and 362 +/- 5 pmol/h per micrograms of hsc70 protein respectively. The EC50 values of hsc70 ATPase for S-peptide, peptides P3a and GT4 are 2, 0.67 and 0.17 mM respectively. On the other hand, the dissociation constants of S-peptide, peptides P3a and GT4 for recombinant hsc70 are 7.6, 13 and 100 microM respectively. Thus peptide GT4 is the only peptide examined for which the binding constant is comparable with the EC50 for stimulation ATPase activity, albeit it has the lowest affinity for hsc70.

A T7-expression system under temperature control could create temperature-sensitive phenotype of target gene in Escherichia coli

2007 ◽  
Vol 68 (3) ◽  
pp. 497-506 ◽  
Author(s):  
Rubing Liang ◽  
Xipeng Liu ◽  
Jianhua Liu ◽  
Qiushi Ren ◽  
Peiji Liang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Temperature Control ◽  
Target Gene ◽  
Expression System ◽  
Temperature Sensitive ◽  
T7 Expression System ◽  
Temperature Sensitive Phenotype
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