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Heterologous expression of membrane proteins: choosing the appropriate host.

ABSTRACT: BACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein.

SUBMITTER: Bernaudat F

PROVIDER: S-EPMC3244453 | biostudies-other | 2011

REPOSITORIES: biostudies-other

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Heterologous expression of membrane proteins: choosing the appropriate host.

Bernaudat Florent F Frelet-Barrand Annie A Pochon Nathalie N Dementin Sébastien S Hivin Patrick P Boutigny Sylvain S Rioux Jean-Baptiste JB Salvi Daniel D Seigneurin-Berny Daphné D Richaud Pierre P Joyard Jacques J Pignol David D Sabaty Monique M Desnos Thierry T Pebay-Peyroula Eva E Darrouzet Elisabeth E Vernet Thierry T Rolland Norbert N

PloS one 20111221 12

<h4>Background</h4>Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein.<h4>Methodology/principal findings</h4>The expression of 20 ...[more]

PMID: 22216205

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Project description:BackgroundWe aimed to investigate the accuracy of different equations in evaluating estimated glomerular filtration rate (eGFR) in a Chinese population with different BMI levels.MethodsA total of 837 Chinese patients were enrolled, and the eGFRs were calculated by three Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equations, three full-age spectrum (FAS) equations and two Modification of Diet in Renal Disease (MDRD) equations. Results of measured GFR (mGFR) by the 99Tcm-diathylenetriamine pentaacetic acid (99Tcm-DTPA) renal dynamic imaging method were the reference standards. According to BMI distribution, the patients were divided into three intervals: below 25th(BMIP25), 25th to 75th(BMIP25-75) and over 75th percentiles (BMIP75).ResultsThe medium BMI of the three BMI intervals were 20.9, 24.8 and 28.9 kg/m2, respectively. All deviations from mGFR (eGFR) were correlated with BMI (p < 0.05). The percentage of cases in which eGFR was within mGFR ±30% (P30) was used to represent the accuracy of each equation. Overall, eGFRFAS_Cr_CysC and eGFREPI_Cr_2009 performed similarly, showing the best agreement with mGFR among the eight equations in Bland-Altman analysis (biases: 4.1 and - 4.2 mL/min/1.73m2, respectively). In BMIP25 interval, eGFRFAS_Cr got - 0.7 of the biases with 74.2% of P30, the kappa value was 0.422 in classification of CKD stages and the AUC60 was 0.928 in predicting renal insufficiency, and eGFREPI_Cr_2009 got 2.3 of the biases with 71.8% of P30, the kappa value was 0.418 in classification of CKD stages and the AUC60 was 0.920 in predicting renal insufficiency. In BMIP25-75 interval, the bias of eGFRFAS_Cr_CysC was 4.0 with 85.0% of P30, the kappa value was 0.501 and the AUC60 was 0.941, and eGFRFAS_Cr_CysC showed balanced recognition ability of each stage of CKD (62.3, 63.7, 68.0, 71.4 and 83.3% respectively). In BMIP75 interval, the bias of eGFREPI_Cr_CysC_2012 was 3.8 with 78.9% of P30, the kappa value was 0.484 the AUC60 was 0.919, and eGFREPI_Cr_CysC_2012 equation showed balanced and accurate recognition ability of each stage (60.5, 60.0, 71.4, 57.1 and 100% respectively). In BMIP75 interval, the bias of eGFRFAS_Cr_CysC was - 1.8 with 78.5% of P30, the kappa value was 0.485, the AUC60 was 0.922. However, the recognition ability of each stage of eGFRFAS_Cr_CysC eq. (71.1, 61.2, 70.0, 42.9 and 50.0% respectively) was not as good as GFREPI_Cr_CysC_2012 equation.ConclusionFor a Chinese population, we tend to recommend choosing eGFRFAS_Cr and eGFREPI_Cr_2009 when BMI was around 20.9, eGFRFAS_Cr_CysC when BMI was near 24.8, and eGFREPI_Cr_CysC_2012 when BMI was about 28.9.

Project description:IntroductionClinicians and patients face a daunting task when choosing the most appropriate probiotic for their specific needs. Available preparations encompass a diverse and continuously expanding product base, with most available products lacking evidence-based trials that support their use. Even when evidence exists, not all probiotic products are equally effective for all disease prevention or treatment indications. At this point in time, drug regulatory agencies offer limited assistance with regard to guidance and oversight in most countries, including the U.S.MethodsWe reviewed the current medical literature and sources on the internet to survey the types of available probiotic products and to determine which probiotics had evidence-based efficacy data. Standard medical databases from inception to June 2018 were searched and discussions with experts in the field were conducted. We graded the strength of the evidence for probiotics having multiple, randomized controlled trials and developed a guide for the practical selection of current probiotic products for specific uses.ResultsWe found the efficacy of probiotic products is both strain-specific and disease-specific. Important factors involved in choosing the appropriate probiotic include matching the strain(s) with the targeted disease or condition, type of formulation, dose used and the source (manufacturing quality control and shelf-life). While we found many probiotic products lacked confirmatory trials, we found sufficient evidence for 22 different types of probiotics from 249 trials to be included. For example, several types of probiotics had strong evidence for the prevention of antibiotic-associated diarrhea [Saccharomyces boulardii I-745, a three-strain mixture (Lactobacillus acidophilus CL1285, L. casei Lbc80r, L. rhamnosus CLR2) and L. casei DN114001]. Strong evidence was also found for four types of probiotics for the prevention of a variety of other diseases/conditions (enteral-feed associated diarrhea, travellers' diarrhea, necrotizing enterocolits and side-effects associated with H. pylori treatments. The evidence was most robust for the treatment of pediatric acute diarrhea based on 59 trials (7 types of probiotics have strong efficacy), while an eight-strain multi-strain mixture showed strong efficacy for inflammatory bowel disease and two types of probiotics had strong efficacy for irritable bowel disease. Of the 22 types of probiotics reviewed, 15 (68%) had strong-moderate evidence for efficacy for at least one type of disease.ConclusionThe choice of an appropriate probiotic is multi-factored, based on the mode and type of disease indication and the specific efficacy of probiotic strain(s), as well as product quality and formulation.Trial registrationThis review was registered with PROSPERO: CRD42018103979.

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Heterologous expression of membrane proteins: choosing the appropriate host.

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Heterologous expression of membrane proteins: choosing the appropriate host.

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