Membrane Protein Production in E. coli for Applications in Drug Discovery

Snijder, H.J.; Hakulinen, Jonna

doi:10.1007/978-3-319-27216-0_5

Cited by 18 publications

(7 citation statements)

References 90 publications

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“…Overexpression of membrane protein in E. coli may lead to toxicity and low yields of the active protein product. Snijder and Hakulinen (2016) described the challenges associated with overexpression of α-helical membrane proteins and different approaches to overcoming these challenges as well as a detailed protocol to express and screen membrane proteins using a His-specific fluorescent probe and fluorescent size-exclusion chromatography. Strategies for the production of soluble recombinant proteins using E. coli were described in another study (Gurramkonda et al, 2018).…”

Section: Escherichia Colimentioning

confidence: 99%

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Tripathi

Shrivastava

2019

Front. Bioeng. Biotechnol.

402

243

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Infectious diseases, along with cancers, are among the main causes of death among humans worldwide. The production of therapeutic proteins for treating diseases at large scale for millions of individuals is one of the essential needs of mankind. Recent progress in the area of recombinant DNA technologies has paved the way to producing recombinant proteins that can be used as therapeutics, vaccines, and diagnostic reagents. Recombinant proteins for these applications are mainly produced using prokaryotic and eukaryotic expression host systems such as mammalian cells, bacteria, yeast, insect cells, and transgenic plants at laboratory scale as well as in large-scale settings. The development of efficient bioprocessing strategies is crucial for industrial production of recombinant proteins of therapeutic and prophylactic importance. Recently, advances have been made in the various areas of bioprocessing and are being utilized to develop effective processes for producing recombinant proteins. These include the use of high-throughput devices for effective bioprocess optimization and of disposable systems, continuous upstream processing, continuous chromatography, integrated continuous bioprocessing, Quality by Design, and process analytical technologies to achieve quality product with higher yield. This review summarizes recent developments in the bioprocessing of recombinant proteins, including in various expression systems, bioprocess development, and the upstream and downstream processing of recombinant proteins.

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Section: Escherichia Colimentioning

confidence: 99%

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Tripathi

Shrivastava

2019

Front. Bioeng. Biotechnol.

402

243

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“…The most exploited bacterial strain for the production of membrane proteins is BL21(DE3), followed by C43(DE3), C41(DE3), pLysS-containing BL21(DE3), BL21(DE3) CodonPlus and Rosetta™ 2(DE3) (Figure 3A). BL21(DE3) cells have been used successfully in the homologous and heterologous production of membrane proteins [48,67]. The majority of these proteins were expressed using the T7 RNA polymerase (T7RNAP)-based promoter system followed by the ara-, T5-and tet-promoters (Figure 3B).…”

Section: Choosing the Right E Coli Expression Systemmentioning

confidence: 99%

Expression of eukaryotic membrane proteins in eukaryotic and prokaryotic hosts

Kesidis

Depping

Lodé

et al. 2020

Methods

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The production of membrane proteins of high purity and in satisfactory yields is crucial for biomedical research. Due to their involvement in various cellular processes, membrane proteins have increasingly become some of the most important drug targets in modern times. Therefore, their structural and functional characterization is a high priority. However, protein expression has always been more challenging for membrane proteins than for soluble proteins. In this review, we present four of the most commonly-used expression systems for eukaryotic membrane proteins. We describe the benefits and drawbacks of bacterial, yeast, insect and mammalian cells. In addition, we describe the different features (growth rate, yield, post-translational modifications) of each expression system, and how they are influenced by the construct design and modifications of the target gene. Cost-effective and fast-growing E. coli is mostly selected for the production of small, simple membrane proteins that, if possible, do not require post-translational modifications but has the potential for the production of bigger proteins as well. Yeast hosts are advantageous for larger and more complex proteins but for the most complex ones, insect or mammalian cells are used as they are the only hosts able to perform all the post-translational modifications found in human cells. A combination of rational construct design and host cell choice can dramatically improve membrane protein production processes.

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“…Capable of performing many PTM; low cost of culture media; industry-scale fermentation Glycosylation pattern different from mammalian; intracellular recovery of large amount of cells may require specific equipment (French-press); high oxygen demand Improved glyco-engineered strains obtained using the GlycoSwitch® technology; wide range of genetic tools, plasmids, strains, and promoters available; the preference for the respiratory growth allow to be cultivated at high cell densities. Plasma membrane composed of phospholipids, sterols (ergosterol), and sphingolipids (inositol) (Gonçalves et al 2013;Laukens et al 2015;Marredy et al 2011;Pedro et al 2015) Insect (Rajesh et al 2011;Proverbio et al 2013;Zheng et al 2014) Other interesting features to be considered when selecting a host: (1) native intracellular localization of the target protein; proteins that function in specific eukaryotic organelles such as mitochondria, chloroplasts, and peroxisomes will generally benefit from expression hosts that possess such organelles (Fernández and Vega 2016); (2) types of lipids of host membranes; hydrophobic mismatch may occur due to differences in lipid bilayer composition and thickness between hosts, as highlighted for the overexpression of eukaryotic MP in bacteria, where the absence of sterols, sphingolipids and poly-unsaturated fatty acids in E. coli bilayers poses additional challenges to their proper folding (Snijder and Hakulinen 2016); (3) Construct size; proteins larger than 120 kDa are difficult to be efficiently expressed in E. coli, and are typically obtained in very low yields, as inclusion bodies or proteolytically degraded (Fernández and Vega 2016).…”

Section: Escherichia Colimentioning

confidence: 99%

Smoothing membrane protein structure determination by initial upstream stage improvements

Pedro

Queiroz

Passarinha

2019

Appl Microbiol Biotechnol

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Membrane proteins (MP) constitute 20-30% of all proteins encoded by the genome of various organisms and perform a wide range of essential biological functions. However, despite they represent the largest class of protein drug targets, a relatively small number high-resolution 3D structures have been obtained yet. Membrane protein biogenesis is more complex than that of the soluble proteins and its recombinant biosynthesis has been a major drawback, thus delaying their further structural characterization. Indeed, the major limitation in structure determination of MP is the low yield achieved in recombinant expression, usually coupled to low functionality, pinpointing the optimization target in recombinant MP research. Recently, the growing attention that have been dedicated to the upstream stage of MP bioprocesses allowed great advances, permitting the evolution of the number of MP solved structures. In this review, we analyse and discuss effective solutions and technical advances at the level of the upstream stage using prokaryotic and eukaryotic organisms foreseeing an increase in expression yields of correctly folded MP and that may facilitate the determination of their three-dimensional structure. A section on techniques used to protein quality control and further structure determination of MP is also included. Lastly, a critical assessment of major factors contributing for a good decision-making process related to the upstream stage of MP is presented.

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Membrane Protein Production in E. coli for Applications in Drug Discovery

Cited by 18 publications

References 90 publications

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Recent Developments in Bioprocessing of Recombinant Proteins: Expression Hosts and Process Development

Expression of eukaryotic membrane proteins in eukaryotic and prokaryotic hosts

Smoothing membrane protein structure determination by initial upstream stage improvements

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