A Potential Method for One-step Purification and Direct Immobilization of Target Protein in Cell Lysate with Magnetic Microbeads

2021 ◽  
pp. 108182
Author(s):  
Mianxing Luo ◽  
Meng Zhang ◽  
Guo Chen ◽  
Jun Zhao ◽  
Hongwei Guo
Keyword(s):  
Potential Method ◽  
Target Protein ◽  
Magnetic Microbeads ◽  
Cell Lysate ◽  
One Step ◽  
Direct Immobilization

Clickable gold-nanoparticles as generic probe precursors for facile photoaffinity labeling application

2021 ◽  
Author(s):  
Kanna Mori ◽  
Kaori Sakurai
Keyword(s):  
Gold Nanoparticles ◽  
Small Molecules ◽  
Photoaffinity Labeling ◽  
Protein Analysis ◽  
Target Protein ◽  
Photoaffinity Probes ◽  
One Step

Clickable photoreactive gold nanoparticles have been developed to facilitate one-step preparation of photoaffinity probes for bioactive small molecules and their application to target protein analysis.

One-step purification of histone deacetylase from Escherichia coli cell-lysate by counter-current chromatography using aqueous two-phase system

2007 ◽  
Vol 1151 (1-2) ◽  
pp. 158-163 ◽  
Author(s):  
Yoichi Shibusawa ◽  
Naoko Takeuchi ◽  
Kanako Tsutsumi ◽  
Shigeru Nakano ◽  
Akio Yanagida ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Histone Deacetylase ◽  
Coli Cell ◽  
Phase System ◽  
Two Phase ◽  
Aqueous Two Phase System ◽  
Cell Lysate ◽  
One Step ◽  
Counter Current

scholarly journals A novel all-in-one strategy for purification and immobilization of β-1,3-xylanase directly from cell lysate as active and recyclable nanobiocatalyst

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Lixi Cai ◽  
Yunmen Chu ◽  
Xin Liu ◽  
Yue Qiu ◽  
Zhongqi Ge ◽  
...  
Keyword(s):  
Immobilized Enzyme ◽  
Free Form ◽  
The Novel ◽  
Initial Activity ◽  
Algae Biomass ◽  
Cell Lysate ◽  
Ph Tolerance ◽  
Versatile Technique ◽  
Reaction Cycles ◽  
Direct Immobilization

Abstract Background Exploring a simple and versatile technique for direct immobilization of target enzymes from cell lysate without prior purification is urgently needed. Thus, a novel all-in-one strategy for purification and immobilization of β-1,3-xylanase was proposed, the target enzymes were covalently immobilized on silica nanoparticles via elastin-like polypeptides (ELPs)-based biomimetic silicification and SpyTag/SpyCatcher spontaneous reaction. Thus, the functional carriers that did not require the time-consuming surface modification step were quickly and efficiently prepared. These carriers could specifically immobilize the SpyTag-fused target enzymes from the cell lysate without pre-purification. Results The ELPs-SpyCatcher hardly leaked from the carriers (0.5%), and the immobilization yield of enzyme was up to 96%. Immobilized enzyme retained 85.6% of the initial activity and showed 88.6% of the activity recovery. Compared with free ones, the immobilized β-1,3-xylanase showed improved thermal stability, elevated storage stability and good pH tolerance. It also retained more than 70.6% of initial activity after 12 reaction cycles, demonstrating its excellent reusability. Conclusions The results clearly highlighted the effectiveness of the novel enzyme immobilization method proposed here due to the improvement of overall performance of immobilized enzyme in respect to free form for the hydrolysis of macromolecular substrates. Thus, it may have great potential in the conversion of algae biomass as well as other related fields.

scholarly journals A novel all-in-one strategy for purification and immobilization of β-1,3-xylanase directly from cell lysate as active and recyclable nanobiocatalyst

2019 ◽  
Author(s):  
Lixi Cai ◽  
Yunmeng Chu ◽  
Xin Liu ◽  
Yue Qiu ◽  
Zhongqi Ge ◽  
...  
Keyword(s):  
Immobilized Enzyme ◽  
Free Form ◽  
The Novel ◽  
Initial Activity ◽  
Algae Biomass ◽  
Cell Lysate ◽  
Ph Tolerance ◽  
Versatile Technique ◽  
Reaction Cycles ◽  
Direct Immobilization

Abstract Background: Exploring a simple and versatile technique for direct immobilization of target enzymes from cell lysate without prior purification is urgently needed. Thus, a novel all-in-one strategy for purification and immobilization of β-1, 3-xylanase was proposed, the target enzymes were covalently immobilized on silica nanoparticles via ELP-based biomimetic silicification and SpyTag/SpyCatcher spontaneous reaction. Thus, the functional carriers that did not require the time-consuming surface modification step were quickly and efficiently prepared. These carriers could specifically immobilize the SpyTag-fused target enzymes from the cell lysate without pre-purification. Results: The ELPs-SpyCatcher hardly leaked from the carriers (0.5%), and the immobilization yield of enzyme was up to 96%. Immobilized enzyme retained 85.6% of the initial activity and showed 88.6% of the activity recovery. Compared with free ones, the immobilized β-1, 3-xylanase showed improved thermal stability, elevated storage stability and good pH tolerance. It also retained more than 70.6% of initial activity after12 reaction cycles, demonstrating its excellent reusability. Conclusions: The results clearly highlighted the effectiveness of the novel enzyme immobilization method proposed here due to the improvement of overall performance of immobilized enzyme in respect to free form for the hydrolysis of macromolecular substrates. Thus, it may have great potential in the conversion of algae biomass as well as other related fields.

scholarly journals Identification of photocrosslinking peptide ligands by mRNA display

2021 ◽  
Author(s):  
Yuteng Wu ◽  
M. Teresa Bertran ◽  
Dhira Joshi ◽  
Sarah Maslen ◽  
Catherine Hurd ◽  
...  
Keyword(s):  
Cyclic Peptides ◽  
Peptide Ligands ◽  
Target Protein ◽  
Rapid Screening ◽  
Mrna Display ◽  
Proof Of Concept ◽  
Cell Lysate ◽  
Protein Ligand Interactions ◽  
Ligand Interactions ◽  
Versatile Technique

Photoaffinity labelling is a promising method for studying protein-ligand interactions. However, obtaining a specific crosslinker can require significant optimisation. We report a novel mRNA display strategy, photocrosslinking-RaPID (XL-RaPID), and exploit its ability to accelerate the discovery of cyclic peptides that photocrosslink to a target of interest. As a proof of concept, we generated a benzophenone-containing library and applied XL-RaPID screening against a model target, the second bromodomain of BRD3. This crosslinking screening gave two optimal candidates that selectively labelled the target protein in cell lysate. Overall, this work introduces direct photocrosslinking screening as a versatile technique for identifying covalent peptide ligands from mRNA display libraries incorporating reactive warheads.

One-step-purification of penicillin G amidase from cell lysate using ion-exchange membrane adsorbers

2013 ◽  
Vol 444 ◽  
pp. 359-364 ◽  
Author(s):  
Andrea Mönster ◽  
Louis Villain ◽  
Thomas Scheper ◽  
Sascha Beutel
Keyword(s):  
Ion Exchange ◽  
Ion Exchange Membrane ◽  
Penicillin G ◽  
Cell Lysate ◽  
One Step ◽  
Membrane Adsorbers ◽  
Exchange Membrane

One-step analysis of protein complexes in microliters of cell lysate

2005 ◽  
Vol 2 (11) ◽  
pp. 833-835 ◽  
Author(s):  
Oda Stoevesandt ◽  
Karsten Köhler ◽  
Rainer Fischer ◽  
Ian C D Johnston ◽  
Roland Brock
Keyword(s):  
Protein Complexes ◽  
Cell Lysate ◽  
One Step

An Interactive Minicomputer Program for the Indexing and Simulation of Electron Diffraction Patterns

1976 ◽  
Vol 34 ◽  
pp. 542-543
Author(s):  
R.P. Goehner ◽  
W.T. Hatfield ◽  
Prakash Rao
Keyword(s):  
Electron Diffraction ◽  
Real Time ◽  
Pattern Analysis ◽  
Flow Diagram ◽  
Hard Copy ◽  
Time Analysis ◽  
Real Time Analysis ◽  
Transmission Electron ◽  
One Step ◽  
Diffraction Patterns

Computer programs are now available in various laboratories for the indexing and simulation of transmission electron diffraction patterns. Although these programs address themselves to the solution of various aspects of the indexing and simulation process, the ultimate goal is to perform real time diffraction pattern analysis directly off of the imaging screen of the transmission electron microscope. The program to be described in this paper represents one step prior to real time analysis. It involves the combination of two programs, described in an earlier paper(l), into a single program for use on an interactive basis with a minicomputer. In our case, the minicomputer is an INTERDATA 70 equipped with a Tektronix 4010-1 graphical display terminal and hard copy unit.A simplified flow diagram of the combined program, written in Fortran IV, is shown in Figure 1. It consists of two programs INDEX and TEDP which index and simulate electron diffraction patterns respectively. The user has the option of choosing either the indexing or simulating aspects of the combined program.

Modulated structures in (Bi,Pb)-Sr-Ca-Cu-O films

1990 ◽  
Vol 48 (4) ◽  
pp. 62-63
Author(s):  
Shozo Ikeda ◽  
Hirotoshi Hayakawa ◽  
Daniel R. Dietderich
Keyword(s):  
Rf Magnetron Sputtering ◽  
Potential Method ◽  
High Tc ◽  
Bscco System ◽  
Heat Treated ◽  
Modulated Structures ◽  
High Tc Phase ◽  
Deposited Films ◽  
Sputtering System

Pb addition makes easier to form the high Tc phase in the BSCCO system. However, Pb easily vaporized at high temperature. A controlled Pb potential method has been applied to grow the high Tc phase in films. Initially, films are deposited on cleaved MgO substrates using an rf magnetron sputtering system. These amorphous as-deposited films are heat treated in a sealed gold capsule along with a large pellet of Pb-added BSCCO. Details of the process and characterization of the films have been reported elsewhere (1). Films trated for 0.5h at 850° C contain mainly the low Tc phase with a small amount of the high Tc phase. Hawever, films treated for 3h at 850°C consist mainly of the high Tc phase. This film is superconductive with a Tc(zero) of 106K. The Pb/Bi ratio of the films, analysed by SEM- EDS, are 0.12 and 0.18 for heat tratment times of 0.5 and 3h, respectively. The present study investigates the modulated structures of these films using HREM.

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