Expression and Purification of Recombinant Proteins in Escherichia coli Tagged with the Metal-Binding Proteins SmbP and CusF3H+

2020 ◽  
pp. 329-344
Author(s):  
Jessica J. Gomez-Lugo ◽  
Bryan D. Santos ◽  
David A. Perez-Perez ◽  
Jorge M. Montfort-Gardeazabal ◽  
Megan M. McEvoy ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Metal Binding ◽  
Binding Proteins ◽  
Expression And Purification

Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF

2016 ◽  
Vol 121 ◽  
pp. 61-65 ◽  
Author(s):  
J. Enrique Cantu-Bustos ◽  
Teresa Vargas-Cortez ◽  
Jose Ruben Morones-Ramirez ◽  
Isaias Balderas-Renteria ◽  
David W. Galbraith ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Metal Binding ◽  
Binding Protein ◽  
Expression And Purification ◽  
Metal Binding Protein

scholarly journals Expression and purification of recombinant proteins from Escherichia coli: Comparison of an elastin-like polypeptide fusion with an oligohistidine fusion

2009 ◽  
Vol 13 (12) ◽  
pp. 3274-3284 ◽  
Author(s):  
Kimberly Trabbic-Carlson ◽  
Li Liu ◽  
Bumjoon Kim ◽  
Ashutosh Chilkoti
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Expression And Purification

scholarly journals Bioaccumulation of Copper Ions by Escherichia coli Expressing Vanabin Genes from the Vanadium-Rich Ascidian Ascidia sydneiensis samea

2003 ◽  
Vol 69 (11) ◽  
pp. 6442-6446 ◽  
Author(s):  
Tatsuya Ueki ◽  
Yasuhisa Sakamoto ◽  
Nobuo Yamaguchi ◽  
Hitoshi Michibata
Keyword(s):  
Escherichia Coli ◽  
Metal Binding ◽  
Binding Proteins ◽  
Copper Ions ◽  
Maltose Binding Protein ◽  
Molar Ratio ◽  
Periplasmic Space ◽  
E Coli ◽  
Genes Encoding

ABSTRACT The genes encoding two vanadium-binding proteins, vanabin1 and vanabin2, from a vanadium-rich ascidian, Ascidia sydneiensis samea, were recently identified and cloned (T. Ueki, T. Adachi, S. Kawano, M. Aoshima, N. Yamaguchi, K. Kanamori, and H. Michibata, Biochim. Biophys. Acta 1626:43-50, 2003). The vanabins were found to bind vanadium(IV), and an excess of copper(II) ions inhibited the binding of vanadium(IV) to the vanabins in vitro. In this study, we constructed Escherichia coli strains that expressed vanabin1 or vanabin2 fused to maltose-binding protein (MBP) in the periplasmic space. We found that both strains accumulated about twenty times more copper(II) ions than the control BL21 strain, while no significant accumulation of vanadium was observed. The strains expressing either MBP-vanabin1 or MBP-vanabin2 absorbed approximately 70% of the copper ions in the medium to which 10 μM copper (II) ions were initially added. The MBP-vanabin1 and MBP-vanabin2 protein expressed in the periplasm bound to copper ions at a copper:protein molar ratio of 8:1 and 5:1, respectively, but MBP did not bind to copper ions. These data showed that the metal-binding proteins vanabin1 and vanabin2 bound copper ions directly and enhanced the bioaccumulation of copper ions by E. coli.

Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP

2019 ◽  
Vol 61 (6) ◽  
pp. 451-460 ◽  
Author(s):  
Bryan D. Santos ◽  
Jose Ruben Morones-Ramirez ◽  
Isaias Balderas-Renteria ◽  
Nestor G. Casillas-Vega ◽  
David W. Galbraith ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Metal Binding ◽  
Binding Protein ◽  
Periplasmic Expression ◽  
Expression In Escherichia Coli ◽  
Metal Binding Protein

scholarly journals High Level Expression and Purification of Recombinant Proteins from Escherichia coli with AK-TAG

PLoS ONE ◽  
2016 ◽  
Vol 11 (5) ◽  
pp. e0156106 ◽  
Author(s):  
Dan Luo ◽  
Caixia Wen ◽  
Rongchuan Zhao ◽  
Xinyu Liu ◽  
Xinxin Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Proteins ◽  
Expression And Purification ◽  
High Level Expression ◽  
High Level

scholarly journals Expression, Purification, and Functional Analysis of Novel RelE Operon fromX. nematophila

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Jitendra Singh Rathore ◽  
Lalit Kumar Gautam
Keyword(s):  
Escherichia Coli ◽  
Functional Analysis ◽  
Recombinant Proteins ◽  
Bacterial Toxin ◽  
Type Ii ◽  
Xenorhabdus Nematophila ◽  
Expression And Purification ◽  
Toxicity Assay ◽  
Neutralization Activity ◽  
E Coli

Bacterial toxin-antitoxin (TA) complexes induce programmed cell death and also function to relieve cell from stress by various response mechanisms.Escherichia coliRelB-RelE TA complex consists of a RelE toxin functionally counteracted by RelB antitoxin. In the present study, a novel homolog of RelE toxin designated as Xn-relE toxin fromXenorhabdus nematophilapossessing its own antitoxin designated as Xn-relEAT has been identified. Expression and purification of recombinant proteins under native conditions with GST and Ni-NTA chromatography prove the existence of novel TA module. The expression of recombinant Xn-relE under tightly regulated ara promoter inE. coliTop 10 cells confirms its toxic nature in endogenous toxicity assay. The neutralization activity in endogenous toxicity assay by Xn-relEAT antitoxin confirms its antidote nature when studying the whole TA operon under ara regulated promoter. This study promotes newly discovered TA module to be regarded as important as other proteins of type II toxin-antitoxin system.

scholarly journals The Small Metal-Binding Protein SmbP Simplifies the Recombinant Expression and Purification of the Antimicrobial Peptide LL-37

Antibiotics ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1271
Author(s):  
David A. Perez-Perez ◽  
Teresa de J. Villanueva-Ramirez ◽  
Adriana E. Hernandez-Pedraza ◽  
Nestor G. Casillas-Vega ◽  
Patricia Gonzalez-Barranco ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Metal Binding ◽  
Recombinant Expression ◽  
Biological Activities ◽  
Production Method ◽  
Biologically Active ◽  
Assay Method ◽  
Expression And Purification ◽  
Cell Lysate

(1) Background: The cathelicidin peptide LL-37 is a prominent molecule with many biological activities, including antimicrobial. Due to its importance, here, we describe the production of LL-37 tagged with SmbP, a relatively new carrier protein that improves the production of recombinant proteins and peptides in Escherichia coli. We present an alternative method for the rapid expression, purification, and antimicrobial evaluation of LL-37, that involves only one purification step. (2) Methods: A DNA construct of SmbP_LL-37 was transformed into E. coli BL21(DE3); after overnight expression, the protein was purified directly from the cell lysate using immobilized metal-affinity chromatography. SmbP_LL-37 was treated with Enterokinase to obtain the free LL-37 peptide. The antimicrobial activity of both SmbP_LL-37 and free LL-37 was determined using the colony forming unit assay method. (3) Results: SmbP_LL-37 was observed in the soluble fraction of the cell lysate; after purification with IMAC, protein gel electrophoresis, and analysis by ImageJ, it showed 90% purity. A total of 3.6 mg of SmbP_LL-37 was produced from one liter of cell culture. SmbP_LL-37 and free LL-37 both showed inhibition activity against Staphylococcus aureus and Escherichia coli. (4) Conclusions: The SmbP fusion protein is a valuable tool for producing biologically-active LL-37 peptide. The production method described here should be of interest for the expression and purification of additional cationic peptides, since it cuts the purification time considerably prior to determination of antimicrobial activity.

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