Importance of the lysine cluster in the translocation of anions through the pyrophosphate specific channel OprO

Piselli, Claudio; Golla, Vinaya Kumar; Benz, Roland; Kleinekathöfer, Ulrich

doi:10.1016/j.bbamem.2022.184086

Cited by 3 publications

(3 citation statements)

References 44 publications

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“…$ CAUTION once you con rmed the presence of your protein via the SDS-PAGE, you can optimize the buffers pH and/or the programming of the machine. 16. Manually, invert the column and clean it with 2 CV of NaCl 2 M, 2 CV of NaOH 1 M and then again with 2 CV of NaCl 2 M at 0.5 mL/min.…”

Section: Puri Cation Timing 3 Hoursmentioning

confidence: 99%

Universal protocol for the isolation of channel forming membrane proteins with the recombinant expression system of E. coli.

Piselli

2023

Preprint

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1 Abstract The heterologous expression, isolation and characterization of pore forming protein is one of the most used strategy for understanding permeability properties of the biological membrane into which they are embedded. Some procedures present in literature are so empirical and long to perform, that the user can easily make mistakes. This protocol will teach you how to quantify and therefore optimize the expression of your favorite protein in E. coli using the BL21Gold(de3)ΔABCF strain. Then with a step-by-step approach, it will tell you how to separate the bacterial compartments according their solubility and then how to extract your protein in its native conformation using detergent. Finally, there is a detailed description on how to improve the purity of your protein via anion exchange chromatography. If you want to study bacterial porins reconstituted in their natural environment, here there is even the description on how to drive them into the outer membrane vesicles where they can be copurified. The protocol is easier and less empirical than the ones of most of the other membrane proteins and represents a solid base for soluble proteins as well (like enzyme or pore forming toxins). Expression time and temperature, concentration of the inducer, nature and concentration of the detergent, incubation time and temperature, purification pH, these are all the parameters that you can troubleshoot according to your results. The expression, extraction and purification will not last longer than a full day each and I have not yet found any protein which could not be isolated thanks to a derivation of this protocol.

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Section: Puri Cation Timing 3 Hoursmentioning

confidence: 99%

Universal protocol for the isolation of channel forming membrane proteins with the recombinant expression system of E. coli.

Piselli

2023

Preprint

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show abstract

“…Also, secondary structures of these channels are very important for their function and three-dimensional architecture and localization in the membrane. For example, lysine residue changed with arginine results in lower conductance in OprP and OprO porins (functional in selective uptake of phosphate molecules) of P. aeroginosa (Piselli et al 2023 ).…”

Section: Introductionmentioning

confidence: 99%

“…Potent drug candidates with a better binding affinity to CarO have been identified with molecular docking and dynamic simulation study (Gopikrishnan and George Priya Doss 2023 ). Understanding the ion mobility across the channels can potentially lead to the development of more effective drug molecules (Piselli et al 2023 ). Interestingly, these outer membrane proteins are induced in high numbers if the bacteria are in contact with lung epithelial cells (Chugani and Greenberg 2007 ; Chevalier et al 2017 ), which results in higher resistance to common antibiotics.…”

Section: Introductionmentioning

confidence: 99%

Microbial membrane transport proteins and their biotechnological applications

Özkan,

Yılmaz,

Ergenekon

et al. 2024

World J Microbiol Biotechnol

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Because of the hydrophobic nature of the membrane lipid bilayer, the majority of the hydrophilic solutes require special transportation mechanisms for passing through the cell membrane. Integral membrane transport proteins (MTPs), which belong to the Major Intrinsic Protein Family, facilitate the transport of these solutes across cell membranes. MTPs including aquaporins and carrier proteins are transmembrane proteins spanning across the cell membrane. The easy handling of microorganisms enabled the discovery of a remarkable number of transport proteins specific to different substances. It has been realized that these transporters have very important roles in the survival of microorganisms, their pathogenesis, and antimicrobial resistance. Astonishing features related to the solute specificity of these proteins have led to the acceleration of the research on the discovery of their properties and the development of innovative products in which these unique properties are used or imitated. Studies on microbial MTPs range from the discovery and characterization of a novel transporter protein to the mining and screening of them in a large transporter library for particular functions, from simulations and modeling of specific transporters to the preparation of biomimetic synthetic materials for different purposes such as biosensors or filtration membranes. This review presents recent discoveries on microbial membrane transport proteins and focuses especially on formate nitrite transport proteins and aquaporins, and advances in their biotechnological applications.

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Outer membrane Proteins-focused Pseudomonas aeruginosa Vaccine Designed using Reverse Vaccinology

Fathi,

Barzegari,

Lotfollahi

et al. 2023

Iran J Med Microbiol

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Importance of the lysine cluster in the translocation of anions through the pyrophosphate specific channel OprO

Cited by 3 publications

References 44 publications

Universal protocol for the isolation of channel forming membrane proteins with the recombinant expression system of E. coli.

Universal protocol for the isolation of channel forming membrane proteins with the recombinant expression system of E. coli.

Microbial membrane transport proteins and their biotechnological applications

Outer membrane Proteins-focused Pseudomonas aeruginosa Vaccine Designed using Reverse Vaccinology

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