Detergents in Membrane Protein Purification and Crystallisation

Anandan, Anandhi; Vrielink, Alice

doi:10.1007/978-3-319-35072-1_2

Cited by 36 publications

(31 citation statements)

References 65 publications

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“…When OmpT IBs were refolded in the absence of the detergent (RC-0), the protein migrated as a single band at 33.5 kDa in an SDS-PAGE gel regardless of heat treatment, indicating that all of the OmpT was unfolded (Figure 2, non-heated and 45 min-heated samples, Lane RC-0). As previously reported, and confirmed here, the presence of the detergent during the refolding process aids in the proper refolding of OmpT from the IBs [33][34][35]. At this point, the apparent refolding efficiencies for RC-31 and RC-10 equal~69% and 32%, respectively, based on the relative intensities of the two bands at 33.5 and 27 kDa for the non-heated samples (Figure 2, non-heated samples, Lane RC-31 and RC-10), a clear indication that excess detergent is essential to achieve higher refolding efficiencies.…”

Section: Autoproteolysis Of Omptsupporting

confidence: 89%

Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding

et al. 2020

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Outer membrane protease (OmpT) is a 33.5 kDa aspartyl protease that cleaves at dibasic sites and is thought to function as a defense mechanism for E. coli against cationic antimicrobial peptides secreted by the host immune system. Despite carrying three dibasic sites in its own sequence, there is no report of OmpT autoproteolysis in vivo. However, recombinant OmpT expressed in vitro as inclusion bodies has been reported to undergo autoproteolysis during the refolding step, thus resulting in an inactive protease. In this study, we monitor and compare levels of in vitro autoproteolysis of folded and unfolded OmpT and examine the role of lipopolysaccharide (LPS) in autoproteolysis. SDS-PAGE data indicate that it is only the unfolded OmpT that undergoes autoproteolysis while the folded OmpT remains protected and resistant to autoproteolysis. This selective susceptibility to autoproteolysis is intriguing. Previous studies suggest that LPS, a co-factor necessary for OmpT activity, may play a protective role in preventing autoproteolysis. However, data presented here confirm that LPS plays no such protective role in the case of unfolded OmpT. Furthermore, OmpT mutants designed to prevent LPS from binding to its putative LPS-binding motif still exhibited excellent protease activity, suggesting that the putative LPS-binding motif is of less importance for OmpT’s activity than previously proposed.

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Section: Autoproteolysis Of Omptsupporting

confidence: 89%

Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding

et al. 2020

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“…One is to modify insoluble proteins at molecular level . The other is the application of detergents or amino acids to increase their solubility before mixing with the crystallization reagent kits . The crystallization reagent kits specifically designed for membrane proteins include: MembFac‐Crystal Screen Lite (Hampton Research); Mem Sys, MemGold, and MemPlus (Molecular Dimensions); as well as MbClass and MbClass II Suites (Qiagen).…”

Section: Overview Of Commercial Screening Kitsmentioning

confidence: 99%

An Analysis on Commercial Screening Kits and Chemical Components in Biomacromolecular Crystallization Screening

Qin

Xie

Zhang

et al. 2019

Crystal Research and Technology

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Obtaining diffraction-quality crystals is still a bottleneck for biomacromolecular structure determination using X-ray diffraction. In practice, the bottleneck mainly comprises two aspects: difficulties in screening and in optimization, and both of them need screening kits. The paper aims at an investigation of the chemical components employed in 86 common commercial screening kits, and comparison with those in the Biological Macromolecules Crystallization Database (BMCD). First, the 86 screening kits are systematically analyzed in terms of design methods, classification, applicable scope, and manufacturing company. Second, the chemical components applied in these screening kits are investigated in detail in terms of the classification and utilization frequency, which are compared with those in BMCD. Third, four main chemical reagents of screening kits including salts, buffers, precipitants, and additives are analyzed in detail. The top ten utilized chemical components are listed. Last, the paper summarizes new progress and further innovation in crystallization condition screening practice and screening kits design. The analysis result would be a valuable reference for rational selection and design of screening kits, and even for personalized screening of a target biomacromolecule, which would provide new ideas to solve the bottleneck problem of biomacromolecular structure determination.

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“…In the absence of—or sometimes complementing—full-length protein structures, useful information may also be obtained from isolated domains in a “divide and conquer” approach to identify structural consequences of mutations or ligands [18, 19]. Once a membrane protein is produced, it must be further purified and reconstituted in solution, generally by replacing most or all of the lipid bilayer with a suitable detergent [20]. Finding a purification and solubilization scheme that preserves structural and functional integrity can be challenging [21], and may require specialized activity assays: substrate binding may be used as a proxy for integrity in membrane transporters, whereas ion flux (e.g.…”

Section: Introductionmentioning

confidence: 99%

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

Howard¹,

Carnevale²,

Delemotte³

et al. 2018

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Ion translocation across biological barriers is a fundamental requirement for life. In many cases, controlling this process-for example with neuroactive drugs-demands an understanding of rapid and reversible structural changes in membrane-embedded proteins, including ion channels and transporters. Classical approaches to electrophysiology and structural biology have provided valuable insights into several such proteins over macroscopic, often discontinuous scales of space and time. Integrating these observations into meaningful mechanistic models now relies increasingly on computational methods, particularly molecular dynamics simulations, while surfacing important challenges in data management and conceptual alignment. Here, we seek to provide contemporary context, concrete examples, and a look to the future for bridging disciplinary gaps in biological ion transport. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

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Detergents in Membrane Protein Purification and Crystallisation

Cited by 36 publications

References 65 publications

Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding

Role of Lipopolysaccharide in Protecting OmpT from Autoproteolysis during In Vitro Refolding

An Analysis on Commercial Screening Kits and Chemical Components in Biomacromolecular Crystallization Screening

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

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