Preparative scale cell-free production and quality optimization of MraY homologues in different expression modes

MraY translocase catalyzes the first committed membrane-bound step of bacterial peptidoglycan synthesis leading to the formation of lipid I. The essential membrane protein therefore has a high potential as target for drug screening approaches to develop antibiotics against gram-positive as well as gram-negative bacteria. However, the production of large integral membrane proteins in conventional cellular expression systems is still very challenging. Cell-free expression technologies have been optimized in recent times for the production of membrane proteins in the presence of detergents (D-CF), lipids (L-CF), or as precipitates (P-CF). We report the development of preparative scale production protocols for the MraY homologues of Escherichia coli and Bacillus subtilis in all three cell-free expression modes followed by their subsequent quality evaluation. Although both proteins can be cell-free produced at comparable high levels, their requirements for optimal expression conditions differ markedly. B. subtilus MraY was stably folded in all three expression modes and showed highest translocase activities after P-CF production followed by defined treatment with detergents. In contrast, the E. coli MraY appears to be unstable after post- or cotranslational solubilization in detergent micelles. Expression kinetics and reducing conditions were identified as optimization parameters for the quality improvement of E. coli MraY. Most remarkably, in contrast to B. subtilis MraY the E. coli MraY has to be stabilized by lipids and only the production in the L-CF mode in the presence of preformed liposomes resulted in stable and translocase active protein samples.     

Comparative analysis and "expression space" coverage of the production of prokaryotic membrane proteins for structural genomics

Membrane proteins comprise up to one-third of prokaryotic and eukaryotic genomes, but only a very small number of membrane protein structures are known. Membrane proteins are challenging targets for structural biology, primarily due to the difficulty in producing and purifying milligram quantities of these proteins. We are evaluating different methods to produce and purify large numbers of prokaryotic membrane proteins for subsequent structural and functional analysis. Here, we present the comparative expression data for 37 target proteins, all of them secondary transporters, from the mesophilic organism Salmonella typhimurium and the two hyperthermophilic organisms Aquifex aeolicus and Pyrococcus furiosus in three different Escherichia coli expression vectors. In addition, we study the use of Lactococcus lactis as a host for integral membrane protein expression. Overall, 78% of the targets were successfully produced under at least one set of conditions. Analysis of these results allows us to assess the role of different variables in increasing "expression space" coverage for our set of targets. This analysis implies that to maximize the number of nonhomologous targets that are expressed, orthologous targets should be chosen and tested in two vectors with different types of promoters, using C-terminal tags. In addition, E. coli is shown to be a robust host for the expression of prokaryotic transporters, and is superior to L. lactis. These results therefore suggest appropriate strategies for high-throughput heterologous overproduction of membrane proteins.     

Expression of G protein coupled receptors in a cell-free translational system using detergents and thioredoxin-fusion vectors

In Escherichia coli and other cell-based expression systems, there are critical difficulties in synthesizing membrane proteins, such as the low protein expression levels and the formation of insoluble aggregates. However, structure determinations by X-ray crystallography require the purification of milligram quantities of membrane proteins. In this study, we tried to solve these problems by using cell-free protein expression with an E. coli S30 extract, with G protein coupled receptors (GPCRs) as the target integral membrane proteins. In this system, the thioredoxin-fusion vector induced high protein expression levels as compared with the non-fusion and hexa-histidine-tagged proteins. Two detergents, Brij35 and digitonin, effectively solubilized the produced GPCRs, with little or no effect on the protein yields. The synthesized proteins were detected by Coomassie brilliant blue staining within 1h of reaction initiation, and were easily reconstituted within phospholipid vesicles. Surprisingly, the unpurified, reconstituted thioredoxin-fused receptor proteins had functional activity, in that a specific affinity binding value of an antagonist was obtained for the receptor. This cell-free translation system (about 1mg/ml of reaction volume for 6-8 h) has biophysical and biochemical advantages for the synthesis of integral membrane proteins.     

Genetic analysis of G protein-coupled receptor expression in Escherichia coli: inhibitory role of DnaJ on the membrane integration of the human central cannabinoid receptor

The overexpression of G protein-coupled receptors (GPCRs) and of many other heterologous membrane proteins in simple microbial hosts, such as the bacterium Escherichia coli, often results in protein mistargeting, aggregation into inclusion bodies or cytoplasmic degradation. Furthermore, membrane protein production is very frequently accompanied by severe cell toxicity. In this work, we have employed a genetic strategy to isolate E. coli mutants that produce markedly increased amounts of the human central cannabinoid receptor (CB1), a pharmacologically significant GPCR that expresses very poorly in wild-type E. coli. By utilizing a CB1 fusion with the green fluorescent protein (GFP) and fluorescence-activated cell sorting (FACS), we screened an E. coli transposon library and identified an insertion in dnaJ that resulted in a large increase in CB1-GFP fluorescence and a dramatic enhancement in bacterial production of membrane-integrated CB1. Furthermore, the dnaJ::Tn5 inactivation suppressed the severe cytotoxicity associated with CB1 production. This revealed an unexpected inhibitory role of the chaperone/ co-chaperone DnaJ in the protein folding or membrane insertion of bacterially produced CB1. Our strategy can be easily adapted to identify expression bottlenecks for different GPCRs or any other integral membrane protein, provide useful and unanticipated mechanistic insights, and assist in the construction of genetically engineered E. coli strains for efficient heterologous membrane protein production.     

An efficient and generic strategy for producing soluble human proteins and domains in E. coli by screening construct libraries

The implementation of generic and efficient technologies for the production of recombinant eukaryotic proteins remains an outstanding challenge in structural genomics programs. We have recently developed a new method for rapid identification of soluble protein expression in E. coli, the colony filtration blot (CoFi blot). In this study, the CoFi blot was used to screen libraries where the N-terminal translation start point was randomized. To investigate the efficiency of this strategy, we have attributed a large number of proteins to this process. In a set of 32 mammalian proteins, we were able to double the success rate (from 34 to 68%) of producing soluble and readily purifiable proteins in E. coli. Most of the selected constructs had their N-termini close to predicted domain borders and the method therefore provides a mean for experimental "domain foot printing." Surprisingly, for most of the targets, we also observed expressing constructs that were close to full-length. In summary this strategy constitutes a generic and efficient method for producing mammalian proteins for structural and functional studies.     

Evaluation of detergents for the soluble expression of alpha-helical and beta-barrel-type integral membrane proteins by a preparative scale individual cell-free expression system

Cell-free expression has become a highly promising tool for the fast and efficient production of integral membrane proteins. The proteins can be produced as precipitates that solubilize in mild detergents usually without any prior denaturation steps. Alternatively, membrane proteins can be synthesized in a soluble form by adding detergents to the cell-free system. However, the effects of a representative variety of detergents on the production, solubility and activity of a wider range of membrane proteins upon cell-free expression are currently unknown. We therefore analyzed the cell-free expression of three structurally very different membrane proteins, namely the bacterial alpha-helical multidrug transporter, EmrE, the beta-barrel nucleoside transporter, Tsx, and the porcine vasopressin receptor of the eukaryotic superfamily of G-protein coupled receptors. All three membrane proteins could be produced in amounts of several mg per one ml of reaction mixture. In general, the detergent 1-myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] was found to be most effective for the resolubilization of membrane protein precipitates, while long chain polyoxyethylene-alkyl-ethers proved to be most suitable for the soluble expression of all three types of membrane proteins. The yield of soluble expressed membrane protein remained relatively stable above a certain threshold concentration of the detergents. We report, for the first time, the high-level cell-free expression of a beta-barrel type membrane protein in a functional form. Structural and functional variations of the analyzed membrane proteins are evident that correspond with the mode of expression and that depend on the supplied detergent.     

Proteomic analysis of the cell envelope fraction of Escherichia coli

We applied proteomics technologies to analyze a membrane preparation of Escherichia coli, wild type strain and of transformants expressing human cytochrome P450s. The proteins were analyzed by two-dimensional electrophoresis and identified by matrix-assisted laser desorption ionization mass spectrometry. The membrane proteins were solubilized with both mild detergents such as CHAPS and strong detergents, such as sodium and lithium dodecyl sulfate, sodium cholate and sodium deoxycholate. In the E. colimembrane fraction, 394 different gene products were identified. Approximately 28% of them were predicted to be integral membrane proteins, of which 100 proteins have been predicted to carry one transmembrane region, ten proteins to carry two, and two proteins to include three transmembrane domains. The remaining are probably membrane-associated and cytosolic proteins. Cytochrome P450s did not enter the immobilized pH gradient strips but were efficiently analyzed in a two-dimensional, two-detergent system. Use of strong solubilizing agents resulted in the detection of about 20 membrane proteins, which were not detected following extraction with mild detergents and chaotropes. The present database is one of the largest for membrane proteins.     

Expression screening,protein purification and NMR analysis of human protein domains for structural genomics

Structural genomics, the determination of protein structures on a genome-wide scale, is still in its infancy for eukaryotes due to the number and size of their genes. Low protein expression and solubility of eukaryotic geneproducts are the major bottlenecks in high-throughput (HTP) recombinant protein production with the E. coli expression systems. To circumvent this problem we decided to focus on separate protein domains. We describe here a fast microtiterplate based, expression and solubility screening procedure, using a combination of in vitro and in vivo expression, and purification with nickel-NTA magnetic beads. All steps are optimized for automatic HTP processing using a liquid handling station. Furthermore, large-scale expression and protein purification conditions are optimized, permitting the purification of 24 protein samples per week. We further show that results obtained from the expression screening can be extrapolated to the production of protein samples for NMR. Starting with 81 cloned human protein domains, in vivo expression was detected in 54 cases, and from 28 of those milligrams of protein were purified. An informative HSQC spectrum was recorded for 18 proteins (22%), half of which were indicative of a folded protein. The success rate and quality of the HSQC spectra suggest that the domain approach holds promise for human proteins.     

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.     

High-throughput screening of soluble recombinant proteins

The aims of high-throughput (HTP) protein production systems are to obtain well-expressed and highly soluble proteins, which are preferred candidates for use in structure-function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96-well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products. All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%). To screen for well-expressed soluble fusion protein, total cell lysates of bacteria culture ( approximately 1.5 mL) were subjected to high-speed centrifugation in a 96-tube format and analyzed by multiwell denaturing SDS-PAGE. Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. The method is well suited for automation and is applicable for the production of large numbers of proteins for genome-wide analysis.     

Protein K: a new major outer membrane protein found in encapsulated Escherichia coli.

The protein composition of purified outer membranes of 47 Escherichia coli strains was examined by sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. Of 33 encapsulated strains, all contained an outer membrane protein distinguishable from previously reported proteins. The 14 non-encapsulated strains with one exception lacked this protein. Because of its apparent association with encapsulation (K antigen) we have named it K protein. The protein was purified nearly to homogeneity by chromatography in the presence of detergents, and its composition was determined. Its amino acid composition does not differ significantly from that reported for protein I, another E. coli major outer membrane protein. Furthermore, the N-terminal amino acid sequence of protein K indicates that it is related to protein I.     

Development and validation of a high throughput system for discovery of antigens for autoantibody detection

An assay employing a panel of tumor-associated antigens has been validated and is available commercially (EarlyCDT®-Lung) to aid the early detection of lung cancer by measurement of serum autoantibodies. The high throughput (HTP) strategy described herein was pursued to identify new antigens to add to the EarlyCDT-Lung panel and to assist in the development of new panels for other cancers. Two ligation-independent cloning vectors were designed and synthesized, producing fusion proteins suitable for the autoantibody ELISA. We developed an abridged HTP version of the validated autoantibody ELISA, determining that results reflected the performance of the EarlyCDT assay, by comparing results on both formats. Once validated this HTP ELISA was utilized to screen multiple fusion proteins prepared on small-scale, by a HTP expression screen. We determined whether the assay performance for these HTP protein batches was an accurate reflection of the performance of R&D or commercial batches. A HTP discovery platform for the identification and optimal production of tumor- associated antigens which detects autoantibodies has been developed and validated. The most favorable conditions for the exposure of immunogenic epitopes were assessed to produce discriminatory proteins for use in a commercial ELISA. This process is rapid and cost-effective compared to standard cloning and screening technologies and enables rapid advancement in the field of autoantibody assay discovery. This approach will significantly reduce timescale and costs for developing similar panels of autoantibody assays for the detection of other cancer types with the ultimate aim of improved overall survival due to early diagnosis and treatment.     

An efficient strategy for high throughput screening of recombinant integral membrane protein expression and stability

Membrane proteins account for about 30% of the genomes sequenced to date and play important roles in a variety of cellular functions. However, determining the three-dimensional structures of membrane proteins continues to pose a major challenge for structural biologists due to difficulties in recombinant expression and purification. We describe here a high throughput pipeline for Escherichia coli based membrane protein expression and purification. A ligation-independent cloning (LIC)-based vector encoding a C-terminal green fluorescence protein (GFP) tag was used for cloning in a high throughput mode. The GFP tag facilitated expression screening in E. coli through both cell culture fluorescence measurements and in-gel fluorescence imaging. Positive candidates from the GFP screening were subsequently sub-cloned into a LIC-based, GFP free vector for further expression and purification. The expressed, C-terminal His-tagged membrane proteins were purified via membrane enrichment and Ni-affinity chromatography. Thermofluor technique was applied to screen optimal buffers and detergents for the purified membrane proteins. This pipeline has been successfully tested for membrane proteins from E. coli and can be potentially expanded to other prokaryotes.     

Inhibition of tobacco etch virus protease activity by detergents

Affinity tags such as polyhistidine greatly facilitate recombinant protein production. The solubility of integral membrane proteins is maintained by the formation of protein-detergent complexes (PDCs), with detergent present at concentration above its critical micelle concentration (CMC). Removal of the affinity tag necessitates inclusion of an engineered protease cleavage site. A commonly utilized protease for tag removal is tobacco etch virus (TEV) protease. TEV is available in a recombinant form (rTEV) and frequently contains its own polyhistidine affinity tag for removal after use in enzymatic digestion. Proteolytic cleavage of the tagged domain is carried out by incubation of the protein with rTEV protease. We have observed that the efficiency of rTEV digestion decreases significantly in the presence of a variety of detergents utilized in purification, crystallization, and other biochemical studies of integral membrane proteins. This reduction in protease activity is suggestive of detergent-induced inhibition of rTEV. To test this hypothesis, we examined the effects of detergents upon the rTEV proteolytic digestion of a soluble fusion protein, alpha(1) platelet activating factor acetylhydrolase (PAFAHalpha(1)). Removal of a hexahistidine amino-terminal affinity tag has been characterized in the presence of 16 different detergents at concentrations above their respective CMCs. Our data indicate that half of the detergents tested reduce the activity of rTEV and that these detergents should be avoided or otherwise accounted for during rTEV digestion of recombinant integral membrane proteins.     

A pedestrian guide to membrane protein crystallization

Membrane protein structural biology is a frontier area of modern biomedical research. Twenty to thirty-five percent of the proteins encoded by an organism's genome are integral membrane proteins. Integral membrane proteins, such as channels, transporters, and receptors, are critical components of many fundamental biological processes. Also, many integral membrane proteins are important in biomedical and biotechnological applications; the majority of drug targets are integral membrane proteins. The sharp increase in the number of membrane protein structures over the last several years gives some indication that this field is poised for rather explosive growth as more and more investigators take on membrane protein projects. The purpose of this brief practical review was to take a snapshot of a field at the onset of its likely exponential growth phase, and to lay out the methods that have worked to date for obtaining membrane protein crystals suitable for structure determination by X-ray crystallography. Many of the successful experimental methods are identical to those used for soluble proteins. The major difference, and a non-trivial difference, is the necessity for inclusion of detergents above the critical micelle concentration in the purified membrane protein solution.     

外源蛋白在大肠杆菌中的表达定位策略

外源基因在大肠杆菌中表达是对基因重组技术的成功应用。外源基因在不同的大肠杆菌表达系统中表达产物可能定位于大肠杆菌空间结构的不同位置:胞质,胞质膜,胞周质,胞外膜和胞外培养基,五种表达定位方式各有其特点和用途 。     

New ligation-independent cloning vectors compatible with a high-throughput platform for parallel construct expression evaluation using baculovirus-infected insect cells

Biomedical research has undergone a major shift in emphasis over the past decade from characterizing the genomes of organisms to characterizing their proteomes. The high-throughput approaches that were successfully applied to sequencing of genomes, such as miniaturization and automation, have been adapted for high-throughput cloning and protein production. High-throughput platforms allow for a multi-construct, multi-parallel approach to expression optimization and construct evaluation. We describe here a series of baculovirus transfer and expression vectors that contain ligation-independent cloning regions originally designed for use in high-throughput Escherichia coli expression evaluation. These new vectors allow for parallel cloning of the same gene construct into a variety of baculovirus or E. coli expression vectors. A high-throughput platform for construct expression evaluation in baculovirus-infected insect cells was developed to utilize these vectors. Data from baculovirus infection expression trials for multiple constructs of two target protein systems relevant to the study of human diseases are presented. The target proteins exhibit a wide variation in behavior and illustrate the benefit of investigating multiple cell types, fusion partners and secretion signals in optimization of constructs and conditions for eukaryotic protein production.     

Membrane protein expression and production: effects of polyhistidine tag length and position

Polyhistidine tags enable the facile purification of proteins by immobilized metal affinity chromatography (IMAC). Both the type and position of purification tags can affect significantly properties of a protein such as its expression level, behavior in solution, and its ability to form suitable samples (esp. suitable crystals for X-ray crystallography). We investigated systematically the effects of polyhistidine tag length and position on many properties related to expression and purification of recombinant integral membrane proteins. Specifically, modified Escherichia coli pET expression vectors were built that placed 6- or 10-histidine tags at the N- or C-termini of the subcloned gene. The E. coli water channel AqpZ was subcloned into this suite of vectors and its expression, purification, solution properties, and yield were characterized. These studies show that: (1) all vectors yield similar expression levels, (2) tag length has a greater effect than tag position upon yield, (3) neither tag length nor position affects significantly detergent solubilization of the protein, (4) the length of the tag affects the oligomerization state of the purified protein, and (5) the tag length and position change chromatographic behavior of the detergent-solubilized protein. In addition, substitution of the lysine codon AAA at the second position, previously shown to have some effect upon soluble protein expression levels, did not have a large effect on AqpZ production. We are currently producing approximately 12 mg of purified AqpZ per liter of shake-flask culture, and preliminary crystals that diffract to approximately 5A resolution have been obtained.