Revolutionizing membrane protein overexpression in bacteria

Schlegel, Susan; Klepsch, Mirjam; Gialama, Dimitra; Wickström, David; Slotboom, Dirk Jan; Gier, Jan‐Willem de

doi:10.1111/j.1751-7915.2009.00148.x

Cited by 59 publications

(55 citation statements)

References 69 publications

(139 reference statements)

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“…Various strategies have been tested to address this issue and obtained optimum expression (9,19,22). Inducible expression systems are one of the most promising approaches for the stable expression of transmembrane proteins.…”

Section: Transformation Of Amb-1 Using Tetracycline-inducible Expressmentioning

confidence: 99%

Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

Yoshino

Shimojo

Maeda

et al. 2010

Appl Environ Microbiol

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Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracyclineinducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracyclineinducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs.Transmembrane proteins play critical roles in cellular metabolism, participating in processes such as ion transport, nutrient uptake, signal transduction, and intercellular communication. As evidence of the essential functions of these proteins, more than half of all drug targets have been shown to be transmembrane proteins, and the analysis of the interactions of transmembrane proteins and their ligands is one of the most promising avenues for the discovery of new drug candidates. As a means of producing sufficient amounts of transmembrane proteins for binding analyses, heterologous protein expression systems have been developed using Escherichia coli (10), yeast (16), insect, and mammalian (4) cells as hosts. Transmembrane proteins generally are expressed at low levels and are extremely hydrophobic, rendering the analysis of interactions with ligands very difficult. In all cases, the analysis of membrane proteins requires a lipid or similar synthetic environment to maintain the native structure and function of the proteins. The purification of transmembrane proteins from cells frequently is time-consuming and typically results in the loss of the proteins' native conformation.Magnetospirillum magneticum AMB-1 synthesizes intracellular nanosized bacterial magnetic particles (BacMPs; 50 to 100 nm); these are surrounded by a lipid bilayer membrane and exhibit strong ferrimagnetism. Functional soluble proteins have been expressed on BacMP surfaces through gene fusion techniques (11,21,24,27) using BacMP membrane proteins (MagA, Mms16, and Mms13) as anchor proteins; this approach permits heterologous proteins to be localized eff...

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Section: Transformation Of Amb-1 Using Tetracycline-inducible Expressmentioning

confidence: 99%

Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

Yoshino

Shimojo

Maeda

et al. 2010

Appl Environ Microbiol

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“…In all cases, the ROC curves were determined using the data set with all 140 TatC loop-swap and point mutations discussed above. The AUC for the C-tail measure (p (7) ) is 0.73, indicating the strong predictive capacity of this measure. However, it is clear that all other measures of integration efficiency fail to offer predictive capacity (yielding AUC values that are within 95% confidence of 0.5).…”

Section: Tatc Topology Features Other Than C-tail Localization Are mentioning

confidence: 99%

“…In this notation, the previously discussed measure of IMP integration based on the C-tail is given by p (7) . Using each of these measures of IMP integration, we obtained ROC curves that compare the simulated integration efficiency with observed experimental expression, and the corresponding AUC values are presented in Fig.…”

Section: Tatc Topology Features Other Than C-tail Localization Are mentioning

confidence: 99%

“…Even among homologous IMP sequences, expression levels can vary widely (1)(2)(3)(4)(5)(6), and the mechanistic basis for this variability is often unclear. Extensive efforts have been committed to identify IMP sequences, expression conditions, and host modifications that yield IMP expression at sufficient levels for further study (7)(8)(9)(10). Despite these efforts, general guidelines for successful overexpression for IMPs are lacking.…”

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confidence: 99%

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Improving membrane protein expression by optimizing integration efficiency

Niesen¹,

Marshall²,

Miller

et al. 2017

Journal of Biological Chemistry

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The heterologous overexpression of integral membrane proteins in Escherichia coli often yields insufficient quantities of purifiable protein for applications of interest. The current study leverages a recently demonstrated link between co-translational membrane integration efficiency and protein expression levels to predict protein sequence modifications that improve expression. Membrane integration efficiencies, obtained using a coarse-grained simulation approach, robustly predicted effects on expression of the integral membrane protein TatC for a set of 140 sequence modifications, including loop-swap chimeras and single-residue mutations distributed throughout the protein sequence. Mutations that improve simulated integration efficiency were 4-fold enriched with respect to improved experimentally observed expression levels. Furthermore, the effects of double mutations on both simulated integration efficiency and experimentally observed expression levels were cumulative and largely independent, suggesting that multiple mutations can be introduced to yield higher levels of purifiable protein. This work provides a foundation for a general method for the rational overexpression of integral membrane proteins based on computationally simulated membrane integration efficiencies. Integral membrane proteins (IMPs)4 play crucial roles in the transport of molecules, energy, and information across the membrane and are an important focus of structural and biophysical studies. However, the production of sufficient levels of IMPs is a limiting factor in their characterization (1). Even among homologous IMP sequences, expression levels can vary widely (1-6), and the mechanistic basis for this variability is often unclear. Extensive efforts have been committed to identify IMP sequences, expression conditions, and host modifications that yield IMP expression at sufficient levels for further study (7-10). Despite these efforts, general guidelines for successful overexpression for IMPs are lacking.Biogenesis of IMPs in Escherichia coli involves multiple steps that are potential bottlenecks for overexpression, including correct targeting to the inner membrane (11, 12), membrane integration (2, 13-17), and folding (18 -21). For a given sequence, understanding how each of these steps affects observed expression levels may lead to improved strategies for IMP overexpression.Previous work indicates that the Sec-facilitated membrane integration step of biogenesis is a limiting factor in the overexpression of the TatC IMP (2). Sequence changes in the C-tail that alter the efficiency of membrane integration efficiency, determined either from coarse-grained (CG) simulations or experimentally, were shown to correlate with experimentally observed IMP expression levels. Further work is necessary to explore the generality of this link and its potential for enabling the rational enhancement of IMP expression.The current study demonstrates the predictive capacity of simulated integration efficiency for experimental expression by examining a wide...

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“…Membrane proteins expression encounters more challenges and difficulty. For the purpose of obtaining membrane proteins, toxicity problem can be reduced by choosing an appropriate host, using cell-free system or using membrane protein variants (Bernaudat et al, 2011;Schlegel et al, 2010). While for the proteins involved in electron transfer, the mentioned general methods for membrane proteins expression are not all suitable here.…”

Section: Besmentioning

confidence: 99%

Metabolic engineering and bio-electrochemical synthesis in Pseudomonas putida KT2440 for the production of p-hydroxybenzoate and 2-ketogluconate

Yu¹

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Bio-based chemicals have drawn research interests due to the need for sustainable development. Aromatics and the derived compounds are an important class of chemicals that are mostly derived from fossil resources. Microbial bio-production can produce many compounds from renewable feedstocks to reduce the current heavy dependency on fossil resources. The aromatic compound para-hydroxybenzoate (PHBA) is used to make parabens and high-value polymers (liquid crystal polymer). Biologically, this chemical can be derived from the shikimate pathway, the central pathway for the biosynthesis of aromatic amino acids in bacteria, plants and fungi and some protozoa. In recent years, the gram-negative soil bacterium Pseudomonas putida is becoming an interesting chassis for industrial biotechnology. The production of PHBA in recombinant P. putida KT2440 was engineered by overexpressing the chorismate lyase Ubic from Escherichia coli and a feedback resistant 3-Deoxy-D-arabinoheptulosonate 7-phosphate synthase (AroG D146N).Additionally, the pathways competing for the substrate chorismate (trpE and pheA) and the PHBA degradation pathway (pobA) were eliminated. Finally, deletion of the glucose metabolism repressor hexR led to an increase in erythrose-4-phosphate and NADPH supply. This resulted in a maximum titre of 1.73 g L -1 and a carbon yield of 18.1 % (C-mol) in a non-optimized fed-batch fermentation. This is to date the highest PHBA concentration produced by P. putida using a chorismate lyase route.Large-scale aerobic fermentations are more expensive due to the limiting gas transfer and lead to substrate loss in the form of CO2, whereas anaerobic processes are easier to scale up and achieve high carbon yield. In the case of aromatics, anaerobic production could be beneficial. Microbial electrosynthesis is an emerging technology for biosynthesis of chemicals and fuels by microorganisms in an anaerobic bio-electrochemical system (BES).These systems can provide electrons to electroactive microbes for reductive production processes in the cathode or drain excessive electrons in oxidative production processes in the anode. P. putida was tested for the first time for its ability to perform anoxic metabolism in BES with ferricyanide as the mediator and anode as the electron sink. A dual-phase fermentation was employed, where the cells grew aerobically in the first phase and then performed an anaerobic oxidation process in the second phase. In this way, P. putida ) with a 9 % lower productivity than that in TB-grown cells. The proteomics analysis suggested the GADH enzyme complex (PP_3382, PP_3383, and PP_3384) overexpression was limited by the subunit PP_3382 biosynthesis. The slight upregulation of PP_3382 in glycerol-grown cells may contribute to its better performance in this condition, suggesting that the aerobic growth condition can be optimized for a better performance in bio-electrochemical production.In summary, using a chorismate lyase route in P. putida is a suitable strategy to produce aromatics. The use of a bio-...

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Revolutionizing membrane protein overexpression in bacteria

Cited by 59 publications

References 69 publications

Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

Improving membrane protein expression by optimizing integration efficiency

Metabolic engineering and bio-electrochemical synthesis in Pseudomonas putida KT2440 for the production of p-hydroxybenzoate and 2-ketogluconate

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