scholarly journals Understanding the yeast host cell response to recombinant membrane protein production

2011 ◽  
Vol 39 (3) ◽  
pp. 719-723 ◽  
Author(s):  
Zharain Bawa ◽  
Charlotte E. Bland ◽  
Nicklas Bonander ◽  
Nagamani Bora ◽  
Stephanie P. Cartwright ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Molecular Mechanisms ◽  
New Drugs ◽  
Host Cells ◽  
Host Cell Response ◽  
Wide Range

Membrane proteins are drug targets for a wide range of diseases. Having access to appropriate samples for further research underpins the pharmaceutical industry's strategy for developing new drugs. This is typically achieved by synthesizing a protein of interest in host cells that can be cultured on a large scale, allowing the isolation of the pure protein in quantities much higher than those found in the protein's native source. Yeast is a popular host as it is a eukaryote with similar synthetic machinery to that of the native human source cells of many proteins of interest, while also being quick, easy and cheap to grow and process. Even in these cells, the production of human membrane proteins can be plagued by low functional yields; we wish to understand why. We have identified molecular mechanisms and culture parameters underpinning high yields and have consolidated our findings to engineer improved yeast host strains. By relieving the bottlenecks to recombinant membrane protein production in yeast, we aim to contribute to the drug discovery pipeline, while providing insight into translational processes.

scholarly journals Membrane-active Polymers: NCMNP13-x, NCMNP21-x and NCMNP21b-x for Membrane Protein Structural Biology

2022 ◽  
Author(s):  
Thi Kim Hoang Trinh ◽  
Claudio Catalano ◽  
Youzhong Guo
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Structural Biology ◽  
Maleic Acid ◽  
Drug Targets ◽  
Design Synthesis ◽  
Native Cell ◽  
Wide Range ◽  
Lipid Particles ◽  
High Resolution Structure

Membrane proteins are a ubiquitous group of bio-macromolecules responsible for many crucial biological processes and serve as drug targets for a wide range of modern drugs. Detergent-free technologies such as styrene-maleic acid lipid particles (SMALP), diisobutylene-maleic acid lipid particles (DIBMALP), and native cell membrane nanoparticles (NCMN) systems have recently emerged as revolutionary alternatives to the traditional detergent-based approaches for membrane protein research. NCMN systems aim to create a membrane-active polymer library suitable for high-resolution structure determination. Herein, we report our design, synthesis, characterization and comparative application analyses of three novel classes of NCMN polymers, NCMNP13-x, NCMNP21-x and NCMNP21b-x. Although each NCMN polymer can solubilize various model membrane proteins and conserve native lipids into NCMN particles, only the NCMNP21b-x series reveals lipid-protein particles with good buffer compatibility and high homogeneity suitable for single-particle cryo-EM analysis. Consequently, the NCMNP21b-x polymers that bring out high-quality NCMN particles are particularly attractive for membrane protein structural biology.

scholarly journals Stress-Responsive Systems Set Specific Limits to the Overproduction of Membrane Proteins in Bacillus subtilis

2009 ◽  
Vol 75 (23) ◽  
pp. 7356-7364 ◽  
Author(s):  
Jessica C. Zweers ◽  
Thomas Wiegert ◽  
Jan Maarten van Dijl
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Drug Targets ◽  
Protein Production ◽  
Cell Envelope ◽  
Regulatory System ◽  
Interesting Candidate ◽  
High Level ◽  
And Function

ABSTRACT Essential membrane proteins are generally recognized as relevant potential drug targets due to their exposed localization in the cell envelope. Unfortunately, high-level production of membrane proteins for functional and structural analyses is often problematic. This is mainly due to their high overall hydrophobicity. To develop new concepts for membrane protein overproduction, we investigated whether the biogenesis of overproduced membrane proteins is affected by stress response-related proteolytic systems in the membrane. For this purpose, the well-established expression host Bacillus subtilis was used to overproduce eight essential membrane proteins from B. subtilis and Staphylococcus aureus. The results show that the σW regulon (responding to cell envelope perturbations) and the CssRS two-component regulatory system (responding to unfolded exported proteins) set critical limits to membrane protein production in large quantities. The identified sigW or cssRS mutant B. subtilis strains with significantly improved capacity for membrane protein production are interesting candidate expression hosts for fundamental research and biotechnological applications. Importantly, our results pinpoint the interdependent expression and function of membrane-associated proteases as key parameters in bacterial membrane protein production.

scholarly journals High Throughput Expression Screening of Arabinofuranosyltransferases from Mycobacteria

Processes ◽  
2021 ◽  
Vol 9 (4) ◽  
pp. 629
Author(s):  
José Rodrigues ◽  
Vanessa T. Almeida ◽  
Ana L. Rosário ◽  
Yong Zi Tan ◽  
Brian Kloss ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Structural Biology ◽  
High Throughput ◽  
Drug Targets ◽  
Large Scale ◽  
Protein Production ◽  
Expression Screening ◽  
E Coli ◽  
Metal Affinity ◽  
Mycobacterial Cell Wall

Studies on membrane proteins can help to develop new drug targets and treatments for a variety of diseases. However, membrane proteins continue to be among the most challenging targets in structural biology. This uphill endeavor can be even harder for membrane proteins from Mycobacterium species, which are notoriously difficult to express in heterologous systems. Arabinofuranosyltransferases are involved in mycobacterial cell wall synthesis and thus potential targets for antituberculosis drugs. A set of 96 mycobacterial genes coding for Arabinofuranosyltransferases was selected, of which 17 were successfully expressed in E. coli and purified by metal-affinity chromatography. We herein present an efficient high-throughput strategy to screen in microplates a large number of targets from Mycobacteria and select the best conditions for large-scale protein production to pursue functional and structural studies. This methodology can be applied to other targets, is cost and time effective and can be implemented in common laboratories.

scholarly journals Membrane recruitment of Atg8 by Hfl1 facilitates turnover of vacuolar membrane proteins in yeast cells approaching stationary phase

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Cheng-Wen He ◽  
Xue-Fei Cui ◽  
Shao-Jie Ma ◽  
Qin Xu ◽  
Yan-Peng Ran ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Stationary Phase ◽  
Molecular Mechanisms ◽  
Multivesicular Body ◽  
Degradation Process ◽  
Vacuolar Membrane ◽  
Yeast Cells ◽  
Membrane Structures ◽  
Membrane Recruitment

Abstract Background The vacuole/lysosome is the final destination of autophagic pathways, but can also itself be degraded in whole or in part by selective macroautophagic or microautophagic processes. Diverse molecular mechanisms are involved in these processes, the characterization of which has lagged behind those of ATG-dependent macroautophagy and ESCRT-dependent endosomal multivesicular body pathways. Results Here we show that as yeast cells gradually exhaust available nutrients and approach stationary phase, multiple vacuolar integral membrane proteins with unrelated functions are degraded in the vacuolar lumen. This degradation depends on the ESCRT machinery, but does not strictly require ubiquitination of cargos or trafficking of cargos out of the vacuole. It is also temporally and mechanistically distinct from NPC-dependent microlipophagy. The turnover is facilitated by Atg8, an exception among autophagy proteins, and an Atg8-interacting vacuolar membrane protein, Hfl1. Lack of Atg8 or Hfl1 led to the accumulation of enlarged lumenal membrane structures in the vacuole. We further show that a key function of Hfl1 is the membrane recruitment of Atg8. In the presence of Hfl1, lipidation of Atg8 is not required for efficient cargo turnover. The need for Hfl1 can be partially bypassed by blocking Atg8 delipidation. Conclusions Our data reveal a vacuolar membrane protein degradation process with a unique dependence on vacuole-associated Atg8 downstream of ESCRTs, and we identify a specific role of Hfl1, a protein conserved from yeast to plants and animals, in membrane targeting of Atg8.

scholarly journals Membrane Protein Stabilization Strategies for Structural and Functional Studies

Membranes ◽  
2021 ◽  
Vol 11 (2) ◽  
pp. 155
Author(s):  
Ekaitz Errasti-Murugarren ◽  
Paola Bartoccioni ◽  
Manuel Palacín
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Membrane ◽  
Protein Stabilization ◽  
New Drugs ◽  
Cell Physiology ◽  
Protein Preparation ◽  
Functional Studies ◽  
Membrane Protein Stability ◽  
Druggable Targets

Accounting for nearly two-thirds of known druggable targets, membrane proteins are highly relevant for cell physiology and pharmacology. In this regard, the structural determination of pharmacologically relevant targets would facilitate the intelligent design of new drugs. The structural biology of membrane proteins is a field experiencing significant growth as a result of the development of new strategies for structure determination. However, membrane protein preparation for structural studies continues to be a limiting step in many cases due to the inherent instability of these molecules in non-native membrane environments. This review describes the approaches that have been developed to improve membrane protein stability. Membrane protein mutagenesis, detergent selection, lipid membrane mimics, antibodies, and ligands are described in this review as approaches to facilitate the production of purified and stable membrane proteins of interest for structural and functional studies.

Database Study on the Expression and Purification of Membrane Proteins

2021 ◽  
Vol 28 ◽  
Author(s):  
Chen-Yan china Zhang ◽  
Shi-Qi Zhao ◽  
Shi-Long Zhang ◽  
Li-Heng Luo ◽  
Ding-Chang Liu ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Expression ◽  
Drug Targets ◽  
Expression System ◽  
Data Bank ◽  
Hek293 Cells ◽  
Expression And Purification ◽  
Beta Barrel ◽  
Membrane Protein Expression

: Membrane proteins are crucial for biological processes, and many of them are important to drug targets. Understanding the three-dimensional structures of membrane proteins are essential to evaluate their bio function and drug design. High-purity membrane proteins are important for structural determination. Membrane proteins have low yields and are difficult to purify because they tend to aggregate. We summarized membrane protein expression systems, vectors, tags, and detergents, which have deposited in the Protein Data Bank (PDB) in recent four-and-a-half years. Escherichia coli is the most expression system for membrane proteins, and HEK293 cells are the most commonly cell lines for human membrane protein expression. The most frequently vectors are pFastBac1 for alpha-helical membrane proteins, pET28a for beta-barrel membrane proteins, and pTRC99a for monotopic membrane proteins. The most used tag for membrane proteins is the 6×His-tag. FLAG commonly used for alpha-helical membrane proteins, Strep and GST for beta-barrel and monotopic membrane proteins, respectively. The detergents and their concentrations used for alpha-helical, beta-barrel, and monotopic membrane proteins are different, and DDM is commonly used for membrane protein purification. It can guide the expression and purification of membrane proteins, thus contributing to their structure and bio function studying.

Glycolipozyme membrane protein integrase (MPIase): recent data

2014 ◽  
Vol 5 (5) ◽  
pp. 429-438 ◽  
Author(s):  
Ken-ichi Nishiyama ◽  
Keiko Shimamoto
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Structure Determination ◽  
Molecular Chaperone ◽  
Molecular Mechanisms ◽  
Cytoplasmic Membrane ◽  
Amino Sugars ◽  
Glycan Chain ◽  
Preprotein Translocation

AbstractA novel factor for membrane protein integration, from the cytoplasmic membrane of Escherichia coli, named MPIase (membrane protein integrase), has recently been identified and characterized. MPIase was revealed to be essential for the membrane integration of a subset of membrane proteins, despite that such integration reactions have been, thus far, thought to occur spontaneously. The structure determination study revealed that MPIase is a novel glycolipid comprising a glycan chain with three N-acetylated amino sugars connected to diacylglycerol through a pyrophosphate linker. As MPIase catalyzes membrane protein integration, we propose that MPIase is a glycolipozyme on the basis of its enzyme-like function. The glycan chain exhibits a molecular chaperone-like function by directly interacting with substrate membrane proteins. Moreover, MPIase also affects the dimer structure of SecYEG, a translocon, thereby significantly stimulating preprotein translocation. The molecular mechanisms of MPIase functions will be outlined.

Meeting report: membrane proteins in health and disease

2002 ◽  
Vol 80 (5) ◽  
pp. v-xi ◽  
Author(s):  
James D Young ◽  
Joseph R Casey ◽  
Reinhart A.F Reithmeier
Keyword(s):  
Membrane Proteins ◽  
Drug Targets ◽  
Ph Regulation ◽  
Meeting Report ◽  
Inherited Disease ◽  
Waste Products ◽  
Wide Range ◽  
Bicarbonate Transporters ◽  
Health And Disease ◽  
Biology Symposium

This article summarizes the scientific presentations made at a Canadian Society of Biochemistry and Molecular & Cellular Biology Symposium on "Membrane Proteins in Health and Diseases" and two satellite meetings on "Bicarbonate Transporters" and "Nucleoside Transporters" held in Banff, Alberta, 20–24 March 2002. Membrane proteins are encoded by about 1/3 of genes and are involved in a wide range of essential functions, including the transport of nutrients, ions, and waste products across biological membranes. Mutations or changes in the expression of these genes cause an equally wide range of diseases. Membrane proteins are also common drug targets or provide drug entry mechanisms. The importance of membrane proteins in biology and medicine was highlighted by the presentations made at this exciting meeting by an international group of experts.Key words: bicarbonate, genomics, inherited disease, nucleosides, organelles, pH regulation, structural biology, trafficking, transporters.

scholarly journals BRANEart: identify stability strength and weakness regions in membrane proteins

2021 ◽  
Author(s):  
Sankar Basu ◽  
Simon S. Assaf ◽  
Fabian Teheux ◽  
Marianne Rooman ◽  
Fabrizio Pucci
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Conformational Changes ◽  
Large Scale ◽  
Protein Structures ◽  
Accurate Method ◽  
Globular Proteins ◽  
Stability Properties ◽  
Overall Stability ◽  
The Stability

AbstractUnderstanding the role of stability strengths and weaknesses in proteins is a key objective for rationalizing their dynamical and functional properties such as conformational changes, catalytic activity, and protein-protein and protein-ligand interactions. We present BRANEart, a new, fast and accurate method to evaluate the per-residue contributions to the overall stability of membrane proteins. It is based on an extended set of recently introduced statistical potentials derived from membrane protein structures, which better describe the stability properties of this class of proteins than standard potentials derived from globular proteins. We defined a per-residue membrane propensity index from combinations of these potentials, which can be used to identify residues which strongly contribute to the stability of the transmembrane region or which would, on the contrary, be more stable in extramembrane regions, or vice versa. Large-scale application to membrane and globular proteins sets and application to tests cases show excellent agreement with experimental data. BRANEart thus appears as a useful instrument to analyze in detail the overall stability properties of a target membrane protein, to position it relative to the lipid bilayer, and to rationally modify its biophysical characteristics and function. BRANEart can be freely accessed from http://babylone.3bio.ulb.ac.be/BRANEart.

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