Mechanisms and significance of tissue-specific MICU regulation of the mitochondrial calcium uniporter complex, Condensed-phase signaling can expand kinase specificity and respond to macromolecular crowding, Academic & Personal: 24 hour online access, Corporate R&D Professionals: 24 hour online access, https://doi.org/10.1016/j.molcel.2022.08.008, Ubiquitination of phosphatidylethanolamine in organellar membranes, Download Hi-res For a comprehensive list, please refer to Puigbo et al. Rluc8 is inserted between B and C helices. Bacteria co-transformed with recombinant proteins and chaperones cloned in independent plasmids are suitable for expression tuning. 6. Polyhistidine tags offer several advantages for protein purification. When devising a project where a purified soluble active recombinant protein is needed (as is often the case), it is invaluable to have means to (i) detect it along the expression and purification scheme, (ii) attain maximal solubility, and (iii) easily purify it from the E. coli cellular milieu. However, two of the drawbacks of silica as a solid support for affinity purification are the limited reagent chemistry that is available and the relatively low efficiency of surface modification. Kapust R. B., Tozser J., Copeland T. D., Waugh D. S. (2002). Load sample onto the cartridge at 0.5mL/min (to decrease the flow rate open the valve on an unused cartridge position on the manifold). This set consists of five plasmids (pACYC derivatives) which allow overexpression of different chaperones or combinations of them: (i) GroES-GroEL, (ii) DnaK/DnaJ/GrpE, (iii) (i) + (ii), (iv) trigger factor, (v) (i) + (iv). Traditional protein pull-down approaches rely on binding of a protein to an affinity resin, and often this is not a very efficient process. Effect of oxygen limitation and medium composition on. To deter the growth of plasmid-free cells, a resistance marker is added to the plasmid backbone. Can I perform a batch purification using the amylose resin? You've created a Promega.com account. Tunable expression can be achieved using the Lemo21(DE3) strain. (A) Activation of AC by both short and long isoforms of Gs was inhibited by GN13. Protein loading is around 2-3nm. HaloTag Technology is compatible with many protein expression systems and can be applied to proteins expressed in E. coli, mammalian cells and cell-free systems. More information and detailed protocols for the HaloTag Mammalian Pull-Down and Labeling System (Cat.# G6500) and HaloTag Mammalian Pull-Down System (Cat.# G6504) are available in Technical Manual #TM342. By contrast, disulfide bond formation in the cytoplasm is rare, maybe because cysteine residues are part of catalytic sites in many enzymes. However, as the -lactamase is continuously secreted, degradation of the antibiotic ensues and in a couple of hours, ampicillin is almost depleted (Korpimaki et al., 2003). The role of specific GTPase family members across diverse human diseases has been widely established by cancer genome sequencing (e.g.. A comprehensive survey of ras mutations in cancer. Learn about our tools that are helping researchers develop diagnostics and vaccines for the SARS-CoV-2 virus. (JM) Structural details of the 3 helices in different G/GDP proteins. Armstrong, R.N. Copyright 2022 Elsevier Inc. except certain content provided by third parties. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. To determine if your protein is located in an inclusion body, perform the lysis step using FastBreak Cell Lysis Reagent, 10X, as described in Technical Manual #TM060. These techniques represent an alternative to conventional chromatography-based purification methods and can save production costs, especially in large-scale settings (Fong and Wood, 2010). GNP and the side chains of S54, T204 and D223 are shown as sticks. Also, the folding pathways that lead to the correct final conformation and stabilization of the proper folded protein may require specific cofactors in the growth media, for example, metal ions (such as iron-sulfur and magnesium) and polypeptide cofactors. Routledge S. J., Hewitt C. J., Bora N., Bill R. M. (2011). The days where kilograms of animal and plant tissues or large volumes of biological fluids were needed for the purification of small amounts of a given protein are almost gone. Fig. Association (t= 0-120 s) and dissociation (t= 120-240 s) cycles of compounds were started by dipping sensors into cyclic peptide solutions and control buffer. Vials were incubated at 37 C at 60rpm in a water bath and taken at designated time points including 0, 480, 1080 and 1440min. Protein Neddylation: beyond cullin-RING ligases. Then the dataset was integrated using the HKL2000 package (. (2009) and we recommend this article to the curious. This approach has a wide availability of tools and methods, but it is beyond the scope of this paper and is addressed elsewhere (Yaman, 1984; Yee and Blanch, 1992; Moulton, 2013). Figure 2 shows a schematic diagram of the MagneHis Protein Purification System protocol. In autoinduction media, a mixture of glucose, lactose, and glycerol is used in an optimized blend. For these reasons, smaller tags with strong solubility enhancing effects are desirable. Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule. Davis G. D., Elisee C., Newham D. M., Harrison R. G. (1999). Identification of candidate substrates for the Golgi Tul1 E3 ligase using quantitative diGly proteomics in yeast. I want to rebind MBP to the amylose column, but the maltose must be removed. (D) Alignment of Gs/GN13 structure (gray) with the structure of Gs/GTPS (green, PDB: 1AZT). If other tubes are used, change volumes accordingly. A novel prophage independent trp regulated lambda PL expression system. Although GN13, GD20, and their analogs are strong Gs binders, with. False discovery rate was set as 5%. However, the performance of the resin is not degraded by one freeze/thaw cycle. Lactose causes induction of the system and this sugar can be used for protein production. Replication and control of circular bacterial plasmids. Fernandez-Castane A., Vine C. E., Caminal G., Lopez-Santin J. Another protein-based stimulus-responsive purification tags are elastin-like polypeptides (ELPs), which consist of tandem repeats of the sequence VPGXG, where X is Val, Ala, or Gly in a 5:2:3 ratio (Meyer and Chilkoti, 1999). Recombinant organisms for production of industrial products. (A)ct-GD20 (B)cpGD20 (GD20-F10L) (C)ct-GD20-F10L. Membrane anchored GDP-bound Gs forms a heterotrimer with G in the resting state. When the Tt is reached, the ELPprotein fusion selectively and reversibly precipitates, allowing for quick enrichment of the recombinant protein by centrifugation (Banki et al., 2005). Finally, we provide a troubleshooting guide that will come in handy when dealing with difficult-to-express proteins. In addition, plasmid loss is prevented thanks to the hsdSB mutation already present in the parental strain (B834) that gave rise to BL21. The anti-ShineDalgarno sequence drives translational pausing and codon choice in bacteria. This process is repeated as many times as desired. Gs is one of the most frequently mutated G proteins in human cancer. Gene toxicity will not be discussed here and the review of Saida et al. Protein crowding mediates membrane remodeling in upstream ESCRT-induced formation of intraluminal vesicles. Prepare the continuous-density sucrose gradient. For this reason, the use of high copy number plasmids for protein expression by no means implies an increase in production yields. For more information, see Technical Manual #TM060. (1970). Cell Lysis: Cells may be lysed using any number of methods including sonication, French press, bead milling, treatment with lytic enzymes (e.g., lysozyme) or use of a commercially available cell lysis reagent such as the FastBreak Cell Lysis Reagent (Cat.# V8571). By creating an account, you confirm that you accept the. Thus, glucose is typically used as a standard for all assays, and our experience shows that glucose gives an identical molar response to the Glc4-based XGO mixture from tamarind within 5% or better. Trademarks. Main characteristics of protein fusion tags. A drawback associated with this assay is interference from diverse reducing substances, notably proteins (which are also routinely quantified using BCA), that can lead to impractically high backgrounds when assaying crude enzyme preparations. The easiest way to increase the amount of available oxygen in shake vessels is to increase shaking speed. Residues that stabilize GDP binding are shown as sticks. Overexpression of trigger factor prevents aggregation of recombinant proteins in. If culture acidification poses a problem, the media can be buffered with phosphate salts at 50 mM. For example, transcription factors play an important role in regulating transcription by binding to specific recognition sites on the chromosome, often at a genes promoter, and interacting with other proteins in the nucleus. Construction of versatile low-copy-number vectors for cloning, sequencing and gene expression in. are Damon Runyon fellows supported by the Damon Runyon Cancer Research Foundation (DRG-[2229-15]; DRG-[2387-30]; DRG-[2281-17]). GD20 is shown as cyan sticks (A)or cartoon (B). To determine if your protein is located in an inclusion body, perform the lysis step using. A combined approach to improving large-scale production of tobacco etch virus protease. Figure 7. 1998; Malloy et al. Interestingly, GN13 modestly increased Gs steady-state GTPase activity (, To explore how GD20 favors Gs/GDP and inhibits GDP dissociation, we solved a structure of the Gs/GDP/GD20 complex. Butt T. R., Edavettal S. C., Hall J. P., Mattern M. R. (2005). ISG15 counteracts Listeria monocytogenes infection. You have successfully reset your password. image, https://doi.org/10.1038/s41586-020-2992-3, https://doi.org/10.1021/acs.chemrev.6b00737, https://doi.org/10.1016/j.molcel.201903033, https://doi.org/10.1016/j.celrep.2015.08.026, https://doi.org/10.1016/j.molcel.2021.03.020, https://doi.org/10.1534/genetics.112.140467, https://doi.org/10.1016/0092-8674(95)90413-1, https://doi.org/10.1007/s00294-019-00982-y, https://doi.org/10.1016/j.devcel.2011.05.015, https://doi.org/10.1534/genetics.111.130286, https://doi.org/10.1111/j.1600-0854.2011.01314.x, https://doi.org/10.1016/j.tibs.2017.09.002, https://doi.org/10.1038/s41594-021-00614-5, https://doi.org/10.1146/annurev-biochem-061516-044916, https://doi.org/10.1016/j.ceb.2019.12.001, https://doi.org/10.1146/annurev-cellbio-092910-154005, https://doi.org/10.1038/s41586-021-03439-w, https://doi.org/10.1038/s41586-021-03566-4, https://doi.org/10.1038/s41579-018-0020-5, https://doi.org/10.1016/s0092-8674(03)00364-7, https://doi.org/10.1016/s0014-5793(02)03622-0, https://doi.org/10.1016/j.tcb.2020.11.011, https://doi.org/10.1016/j.cell.2019.12.005, https://doi.org/10.1016/S0968-0004(03)00113-0, https://doi.org/10.1016/j.molcel.2011.02.035, https://doi.org/10.1038/s41422-018-0017-7, https://doi.org/10.1016/j.jmb.2005.03.007, https://doi.org/10.1016/S0014-5793(99)00025-3, https://doi.org/10.1074/mcp.M800372-MCP200, https://doi.org/10.1007/s11033-014-3486-x, For academic or personal research use, select 'Academic and Personal', For corporate R&D use, select 'Corporate R&D Professionals'. Like other parental B strains, BL21 cells are deficient in the Lon protease, which degrades many foreign proteins (Gottesman, 1996). Mechanisms of plasmid stable maintenance with special focus on plasmid addiction systems. To determine whether active-state binders inhibit Gs activity, we assayed the ability of Gs to activate its effector, AC (. Membrane bending by protein phase separation. Plasmid copy number control: an ever-growing story. The oriS origin and its control elements maintain pETcoco at one copy per cell (Wild et al., 2002). Designer ribozymes: Programming the tRNA specificity into flexizyme. Production of soluble eukaryotic recombinant proteins in is favoured in early log-phase cultures induced at low temperature. (J) Activation of AC by GNP-bound WT Gs or GNP-bound Gs oncogenic mutant Q227L were inhibited by GN13 but not GD20. Genetic recombination and complementation between bacteriophage T7 and cloned fragments of T7 DNA. Final elution is best achieved by transferring the HisLink Resin to a column to elute the protein in fractions. The Mg, (F) Binding kinetics of GD20 to WT Gs/GDP were quantified using BLI. Sezonov G., Joseleau-Petit D, DAri R. (2007). The S-directed VLP-tEV fusions were quantified by HiBiT:LgBiT complementation into nanoluciferase, measured by luminometry (Fig. 1975; Lonnerdal and Keen, 1982). Mean SD, n= 3. The trfA gene is on the same vector and is under control of the araPBAD promoter, so copy number can be controlled by arabinose (Wild et al., 2002). (A) GN13 and GD20 are class-specific Gs inhibitors. There was an issue verifying your email address. That being said, thanks to the efforts of the scientific community, the general methods available in the literature are no longer anecdotal and can be used systematically. Two-tailed unpaired t tests (data after 5min). Given their function, it is not surprising that efforts to inhibit IB formation were directed to the co-expression of individual or sets of molecular chaperones (Caspers et al., 1994; Nishihara et al., 2000; de Marco et al., 2007). Thus, after cell division, plasmid-free bacteria die. GD20 showed high nucleotide-state selectivity for the GDP-bound Gs (, Video S1. Release of proteins and peptides from fusion proteins using a recombinant plant virus proteinase. Snapshot of Activated G Proteins at the Membrane: The G, To explain GD20 G selectivity, we compared our Gs/GD20 structure with GDP-bound G13, Gq, and Gi (, GPCR signaling releases GTP-bound G and free G to engage their own effectors for downstream signaling. The structure and function of deubiquitinases: lessons from budding yeast. (1988). Contact your local subsidiary or distributor. (2003). There was an issue resetting your password. Many of the approaches described in this review will fail miserably in a lot of cases. Cells were transfected 2AR (100ng), G1-C Venus (25ng), G2-N Venus (25ng), GIRK4-NLuc (100ng). Save 20% On GloMax Instruments! Proteins expressed in bacterial cells may be present in insoluble inclusion bodies. Another gene missing from the genome of the ancestors of BL21 is the one coding for the outer membrane protease OmpT, whose function is to degrade extracellular proteins. S.A.D., Q.H., R.W., H.P., H.S., and K.M.S. 2000; Wada et al. Mean SD, n= 3. GN13-E3Q was chosen to simplify chemical synthesis of biotinylated cyclic peptides. When preparing cell lysates from high-density cultures, adding DNase and RNase (concentrations up to 20g/ml) will reduce the lysate viscosity and aid purification. Menart V., Jevsevar S., Vilar M., Trobis A., Pavko A. Blommel P. G., Becker K. J., Duvnjak P., Fox B. G. (2007). A multipurpose cloning system. Practical considerations in refolding proteins from inclusion bodies. Being a workhorse organism, these strategies arose thanks to the wealth of knowledge about its physiology. More detailed information is available in Technical Manual #TM249. Not surprisingly, increasing the amount of peptone or yeast extract leads to higher cell densities (Studier, 2005). Homologous residues in other G proteins are labeled with different colors. GST pull-down assays use a GST-fusion protein (bait) bound to glutathione (GST)-coupled particles to affinity-purify any proteins that interact with the bait from a pool of proteins (prey) in solution. Increasing the yield of soluble recombinant protein expressed in. By contrast, dose-dependent expression when using IPTG as inducer is not possible since IPTG can enter the cell by active transport through the Lac permease or by permease-independent pathways (Fernandez-Castane et al., 2012). Regulation of lac operon expression: reappraisal of the theory of catabolite repression. These applications involve both manual and automated systems that operate under positive or negative pressure (e.g., FPLC and vacuum systems, respectively). (F and H) Binding of GD20 (F)or cpGD20 (H)to Gs, G(i/13) and Gi were quantified using BLI. Systems that respond to physical signals (e.g., temperature or pH) are also available (Goldstein and Doi, 1995). The advantages of batch purification are: 1) less time is required to perform the purification; 2) large amounts of lysate can be processed; and 3) clearing the lysate prior to purification is not required. Once you have determined the volume of settled resin required, precalibrate this amount directly in the column by pipetting the equivalent volume of water into the column and marking the column to indicate the top of the water. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]. All of this new evidence in translational control mechanisms poses a challenge in the rational design of synthetic genes. The main characteristics of the tags mentioned in this section are outlined on Table Table11. However, something to keep in mind is that many are specialty strains that are used in specific situations. GN13-E3Q-Biotin: HRMS (ESI): Calcd for (C, cpGD20 (GD20-F10L): HRMS (ESI): Calcd for (C, All of the curves in Figuresexcept those from the BLI experiments were fitted by GraphPad Prism. For example, a 1g/L xyloglucan solution contains just below 1mM cleavage sites (assuming an approximate molar mass of an XGO repeat of 1000Da, and cleavage at each unbranched Glc, typical for most endo-xyloglucanases), which means that initial rates should be measured at less than 100M produced reducing ends or lower. The AraC protein has the dual role of repressor/activator. This standard BCA assay can be used to determine the protein concentrations of each homogenized sample. A superfamily of protein tags: ubiquitin, SUMO and related modifiers. The biochemical features of different tags influence the stability, solubility and expression of proteins to which they are attached (Stevens et al. Vera A., Gonzalez-Montalban N., Aris A., Villaverde A. Chloramphenicol acetyltransferase: enzymology and molecular biology. (L) Illustration of inactive-state binders inhibiting PPI between Gs/GDP and G(C68S). In addition, the system can be used with automated liquid-handling platforms for high-throughput applications. Revealing the Activity of Trimeric G-proteins in Live Cells with a Versatile Biosensor Design. Immobilized metal ion affinity adsorption and immobilized metal ion affinity chromatography of biomaterials. Scale-up and optimization of the low-temperature inducible cspA promoter system. MBP6-TEV-Paramyosin-Sal is a positive control for TEV Protease cleavage. 1996). If lactose and glucose are present, expression from the lac promoter is not fully induced until all the glucose has been utilized. Mechanisms of deubiquitinase specificity and regulation. (A) Illustration of Gs/G PPI inhibitors acting as dual-effect G protein modulators in cells. Cyclic peptide loading is around 0.20.3nm. Sahdev S., Khattar S. K., Saini K. S. (2008). international site. The particles are easily and efficiently separated from supernatants using a magnetic stand without centrifugation, increasing reproducibility and reducing sample loss. The https:// ensures that you are connecting to the While this promising technology has been proved successful in large-scale fermentors (Voss and Steinbuchel, 2006; Peubez et al., 2010), expression systems based on plasmid addiction are still not widely distributed. Thank you for verifying your email address. Efficient protection and isolation of ubiquitylated proteins using tandem ubiquitin-binding entities. As a result, DNA methylation and degradation is disrupted. GSTGSTMCSHRV 3C Protease HRV 3C Protease45 Crystal Structure of the Catalytic Domains of Adenylyl Cyclase in a Complex with Gs GTPS. Carefully load the sample prepared in step 4 on top of the sucrose gradient and centrifuge at 100,000xg for 16h at 4C. We recommend using the manual protocol as a guide to develop protocols for automated workstations. Proteins are biological macromolecules that maintain the structural and functional integrity of the cell, and many diseases are associated with protein malfunction. Add 250L BCA solution to 250L of glucose standard or enzyme reaction. Accepted: Mean SD, n= 2. Fill out ourTechnical Support Form, will also be available for a limited time. This led to the erroneous belief that within each cell, the level of recombinant protein synthesis can be manipulated at will. 16h after drug treatment at 37C with 5% CO, The plasmids encoding G1-C Venus, G2-N Venus, and GIRK4-NLuc were gifts from Dr. Roderick MacKinnon. How many times can I use the amylose column? Strategies for overcoming common problems during recombinant protein expression in E. coli. Structural basis for nucleotide exchange in heterotrimeric G proteins. Some proteins require small molecules or prosthetic groups to acquire their final folded conformation. The following proteins were prepared for the AC assay, the radioactivity assay, and the steady-state GTPase assay: AHMSKTEDQRNEEKAQREANKKIEKQLQKDKQVYRATHRLLLLGAGESGKSTIVKQMRILHVNGFNGDSEKATKVQDIKNNLKEAIETIVAAMSNLVPPVELANPENQFRVDYILSVMNVPDFDFPPEFYEHAKALWEDEGVRACYERSNEYQLIDCAQYFLDKIDVIKQADYVPSDQDLLRCRVLTSGIFETKFQVDKVNFHMFDVGGQRDERRKWIQCFNDVTAIIFVVASSSYNMVIREDNQTNRLQEALNLFKSIWNNRWLRTISVILFLNKQDLLAEKVLAGKSKIEDYFPEFARYTTPEDATPEPGEDPRVTRAKYFIRDEFLRISTASGDGRHYCYPHFTCAVDTENIRRVFNDCRDIIQRMHLRQYELL, Adenylyl cyclase C1 domain purification: Residues D628 and S645 in the C1 domain (residues 443-659) of mouse ADCY5 (adenylyl cyclase V) were mutated to glutamic acid and arginine, respectively, resulting a sequence that is the same as the C1 domain of human ADCY5 (residues 442-658). Codon optimization can improve expression of human genes in. Please try again or contact Customer Service. Loading the digest onto NEBExpress Ni Resin sequesters both the MBP-tag and TEV Protease, thereby isolating the target protein in the column flow through. Flush resins with 7mL of ACN at a flow rate of approximately 5mL/min. The specific activity is described as the amount of epoxypropane produced in 1min by 1mg of protein (mol-epoxypropane min1mg-protein1). At higher concentrations of the sugar L-rhamnose, more T7 lysozyme is produced, less active T7 RNAP is present in the cell and less recombinant protein is expressed. Rais-Beghdadi C., Roggero M. A., Fasel N., Reymond C. D. (1998). To save your cart and view previous orders, sign in to your NEB account. The content on this site is intended for healthcare professionals. In comparison studies, GST showed the poorest solubility enhancement capabilities (Hammarstrom et al., 2006; Bird, 2011). INCA: synonymous codon usage analysis and clustering by means of self-organizing map. At the moment of full synthesis of the recombinant protein, depletion of low-abundance tRNAs occurs. 2xYT, TB (Terrific Broth) and SB (Super Broth) media recipes are available elsewhere and have been shown to be superior to LB for reaching higher cell densities (Madurawe et al., 2000; Atlas, 2004; Studier, 2005). Beyond de-foaming: the effects of antifoams on bioprocess productivity. The gene of residues 2-354 of human Gi1 (GNAI1, accession number in PubMed: NP_002060.4) with a stop codon at its end was cloned into the NdeI/XhoI site of a modified pET15b vector, in which a Drice cleavage site (AspGluValAspAla) and an Avi tag were inserted at the N-terminus. A ubiquitin-like system mediates protein lipidation. Mean SD, n= 3. Mean SD, n= 3. The HisLink Resin enables efficient capture and purification of bacterially expressed polyhistidine-tagged proteins. Different subtypes of plasmid-addiction systems exist according to their principle of function: (i) toxin/antitoxin-based systems, (ii) metabolism-based systems, and (iii) operator repressor titration systems (Kroll et al., 2010). Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. The concentrations of pMMO can be determined by the bicinchoninic acid (BCA) assay. The transformed, G purification: Human G1 with a hexahistidine tag at its N terminus and human G2(C68S) were cloned into pFastBac Dual expression vector. Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. What is the minimum size of a fragment that can be cloned into pMAL and expressed fused to MBP? For detailed protocols on use of the HaloTag Mammalian Protein Detection and Purification System, see Technical Manual, TM348. it is conjugated mainly to phospholipids. Hydrolytic activity on polysaccharides, xyloglucan included, is best quantified using assays which assay the generation of new reducing-end groups. Erlin-2 and ACSL4 (acyl-CoA synthetase long chain family member 4) are MAM-resident proteins. Stable expression clones and auto-induction for protein production in. You take your gene of interest, clone it in whatever expression vector you have at your disposal, transform it into the host of choice, induce and then, the protein is ready for purification and characterization. A study reveals that activation of Toll/interleukin-1 receptor signalling is an important mechanism for boosting plant defence during pattern-triggered immunity. Recognition of the activated states of G13 by the rgRGS domain of PDZRhoGEF. GSTGSTMCSHRV 3C Protease HRV 3C Protease45 For this reason, the addition of an antifoaming agent is recommended, although it was shown that antifoams can affect the growth rate of several microorganisms and the yield of recombinant protein (Routledge et al., 2011; Routledge, 2012). Weickert M. J., Pagratis M., Glascock C. B., Blackmore R. (1999). Hexa-histidin tag position influences disulfide structure but not binding behavior of in vitro folded N-terminal domain of rat corticotropin-releasing factor receptor type 2a. Please contact Customer Service to unlock your account. LaVallie E. R., Diblasio E. A., Kovacic S., Grant K. L., Schendel P. F., Mccoy J. M. (1993). Analysis of protein:protein interactions often requires straightforward methods for immobilizing proteins on solid surfaces in proper orientations without disrupting protein structure or function. Akimitsu Miyaji, in Methods in Enzymology, 2011. 2022. Interchangeable functionality of the HaloTag protein tag. The protein may still be of bad quality; i.e., it does not have the activity it should. For the calculation of yield of pMMO purification, propene epoxidation is used for the pMMO assay. The data represent one measurement. Costa S. J., Almeida A., Castro A., Domingues L., Besir H. (2013). Peptides are purified to optimize the efficiency of the succeeding labeling with TMT reagents. The choice of the host cell whose protein synthesis machinery will produce the precious protein will initiate the outline of the whole process. P = prey protein, M = MagneGST Particle. So, even though the final protein yield can be controlled, the amount of protein per cell is widely variable, with cells producing massive amounts of protein and others not producing any protein at all. GPC3-Unc5 receptor complex structure and role in cell migration, Quorum-sensing- and type VI secretion-mediated spatiotemporal cell death drives genetic diversity in Vibrio cholerae, Selection of cyclic peptides that bind to the active or inactive state of Gs, RaPID selection of state-selective Gs binding cyclic peptides, RaPID selection of state-selective Gs binding cyclic peptides, related to, Active-state binding cyclic peptide GN13 blocks Gs-mediated AC activation, Gs active-state inhibitor GN13 inhibits Gs-mediated adenylyl cyclase activation, Gs active-state inhibitor GN13 inhibits Gs-mediated adenylyl cyclase activation, related to, The crystal structure of GppNHp-bound Gs in complex with GN13, GN13 specifically inhibits Gs through binding to a crystallographically defined pocket, related to, The crystal Structure of GppNHp-bound Gs in complex with GN13, Structural basis for the nucleotide-state selectivity of GN13, Inactive-state binding cyclic peptide GD20 is a Gs specific guanine nucleotide dissociation inhibitor, GN13 and GD20 modulate Gs GTPase activity in a Gs-specific manner, related to, The crystal structure of GDP-bound Gs in complex with GD20, GD20 specifically inhibits Gs through binding to a crystallographically defined pocket, related to, Structural basis for the nucleotide-state selectivity and biochemical activity of GD20, eyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI0NDA1N2JhOTQyNzE5MTZmNDgyZDFlMzAyNDIwMzk1YSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjcwODI0ODk0fQ.h_EpPjZe86d2cHS57hh5QM8betZo4TQ0UyJTXrjW9I4twwSQF5eE_JeDtApZFHuH5e3xJNB5uo9VrTuwwLmIoIs8XW7Zw-Uqqq1r2uAFloqA2M1ZhP3h59F4W7YDSR0U8BAfJ0U9ALFvN5uSt-a5Eow-_3voqw8KhRgTdfkrxj98ePKABgq6sIKOJDxlP3t7jCAt-8-UCYkyD6emjz4iwpbcB0ovi_2bpcOhI-akIU00n4ExVL9ZfFNyqmpXPbDd4kncnfEbQfbNI501rlCNrk_lIlQNT2jHWif3K0AqCTamXDZN02tIp1Mijfcm36wiqJ9cPTVdziSF0aLzzfXqsQ, G protein class-specificity of GN13 and GD20, G protein class-specificity of GN13 and GD20, related to, A cell-permeable GD20 analog, cpGD20, is a dual-effect G protein modulator, A cell-permeable GD20 analog, cpGD20, is a dual-effect G protein modulator, related to, Data collection and structure determination, Characterization data for cyclic peptides, https://doi.org/10.1016/j.cell.2022.09.019, State-selective modulation of heterotrimeric Gs signaling with macrocyclic peptides, View Large Strategies for efficient production of heterologous proteins in. Most protocols call for diluting a saturated overnight preculture (dilution factor 1/100) into the larger culture (Sivashanmugam et al., 2009). Madurawe R. D., Chase T. E., Tsao E. I., Bentley W. E. (2000). (B) GN13 did not directly inhibit the intrinsic or forskolin-mediated AC activity in the absence of Gs. Cyanogen bromide cleavage of proteins in salt and buffer solutions. For a 3min reaction, the amount of produced epoxypropane is determined by flame-ionized detector-gas chromatography. The principle of this technique is quite similar to the Lowry protein assay, which is one of the alternative protein quantification methods. (A) Gs steady-state GTPase activity was modulated by GN13 and GD20. The one amino acid-one codon strategy disregards factors other than codon rarity that influence protein expression levels. DNA sequences of cyclic peptide binders from the R4 pools were quantified and identified by qPCR and NGS. (K) GD20 inhibited PPI between Gs/GDP and G(C68S). (D) Alignment of Gs/GD20 complex structure (gray) with the structure of Gs/GTPS (green, PDB: 1AZT) in the switch II/3 pocket. Studies on aminoglycoside antibiotics: enzymic mechanism of resistance and genetics. S2, A and B) . A solubility tag, maltose-binding protein (MBP), was followed by a tobacco etch virus protease (TEV) cleavage site, GFP at the C terminus and non-cleavable His 6 tags at both ends. The target protein yield can be up to 100 mg/L, with typical yields in the range of 1040 mg/L. Steps 17 from the MAM isolation protocol are done to obtain P2 (the crude membrane fraction). In this context, it is worth remembering the rule of thumb that initial-rate enzyme kinetic measurements must be performed with less than 10% substrate conversion. When the protein of interest cannot be detected through a sensitive technique (e.g., Western blot) or it is detected but at very low levels (less than micrograms per liter of culture), the problem often lies in a harmful effect that the heterologous protein exerts on the cell (Miroux and Walker, 1996; Dumon-Seignovert et al., 2004). This deficiency may lead to amino acid misincorporation and/or truncation of the polypeptide, thus affecting the heterologous protein expression levels (which will be low at best) and/or its activity (Gustafsson et al., 2004). Nb35 is shown as cartoon. Modulation of ColE1-like plasmid replication for recombinant gene expression. Hudson, M.E. A peptide tag must be added to the fusion partner-containing protein if an affinity chromatography step is needed in the purification scheme. (B) GDP dissociation from Gs in the presence (red) or absence (black) of 10M GN13 were measured. Production of recombinant proteins by high cell density culture of, Secretory and extracellular production of recombinant proteins using. (D) Structure of the resynthesized cyclic peptide GN13. Recently, the 8-kDa calcium binding protein Fh8 from the parasite Fasciola hepatica was shown to be as good as or better than the large tags in terms of solubility enhancement. If structural or biochemical studies on the recombinant protein are needed, then the fusion partner must be eliminated from the recombinant protein. del Solar G., Giraldo R., Ruiz-Echevarria M. J., Espinosa M., Diaz-Orejas R. (1998). Physiological pH and acidic phospholipids contribute to substrate specificity in lipidation of Atg8. Figure 5. Another solution could be to remove the protein from the cell. Different HaloTag Ligands with different functionalities are available to eliminate the need to design and create a new expression construct. in which Y is the radioactivity (Counts per minute) of the sample at time X (minutes), and Y0 is the calculated radioactivity of the sample at the time point 0. G/GDP is a functional OFF switch: it tightly reassociates with G, masking effector-binding surfaces on both Gs and G (. In E. coli, cysteine oxidation takes places in the periplasm, where disulfide bonds are formed in disulfide exchange reactions catalyzed by a myriad of enzymes, mainly from the Dsb family (Messens and Collet, 2006). To ensure a maximum library diversity at the initial stage of selection, negative selection was not included in the first round of selection. Cultured mammalian cells might offer a better option for producing properly folded and functional mammalian proteins with appropriate post-translational modifications (Geisse et al. Schematic diagram of the MagZ Protein Purification System. His 6 MBP tag was removed by TEV protease cleavage overnight at 4 C with gentle shaking. Its use as a cell factory is well-established and it has become the most popular expression platform. Even after careful selection of plasmid and host, it cannot be predicted if the protein will be obtained in high amounts and in a soluble active form. Self-cleavable stimulus responsive tags for protein purification without chromatography. (I) Close-up view of Gs nucleotide binding pocket in our Gs/GD20 complex structure. tRNA genes rapidly change in evolution to meet novel translational demands. Moreover, the recombinant proteins maintained their solubility after tag removal (Costa et al., 2013). Epstein W., Rothman-Denes L. B., Hesse J. Codon bias arises when the frequency of occurrence of synonymous codons in the foreign coding DNA is significantly different from that of the host. Protein fusion tags are used to aid expression of suitable levels of soluble protein as well as purification. Jorge Montesinos, Estela Area-Gomez, in Methods in Cell Biology, 2020. Small-scale screens can be performed in 2-ml tubes or 96-well plates (Shih et al., 2002). High-throughput screening of soluble recombinant proteins. Welch M., Govindarajan S., Ness J. E., Villalobos A., Gurney A., Minshull J., et al. (1997) Structure, catalytic mechanism, and evolution of the glutathione transferases. Info@neb.com, This product is a direct replacement for NEB #E8200, pMAL Protein Fusion and Purification System. These tags undergo an inverse phase transition at a given temperature of transition (Tt). We are then using a bicinchoninic acid (BCA) assay to determine the concentration of peptides in the samples to adjust the sample input for the consequent phosphopeptide enrichment. TORC1 regulates vacuole membrane composition through ubiquitin- and ESCRT-dependent microautophagy. An official website of the United States government. Learn more Recruitment and organization of ESCRT-0 and ubiquitinated cargo via condensation. The history of the BL21 and BL21(DE3) strains was beautifully documented in Daegelen et al. To identify G protein specificity determinants of both cyclic peptides, we aligned G sequences at the cyclic peptide binding interfaces (. cpGD20 was nearly 100-fold more selective for Gs than Gi. Chant A., Kraemer-Pecore C. M., Watkin R., Kneale G. G. (2005). The signal sequence of disulfide isomerase I (DsbA) has been used to target recombinant proteins to the periplasm via the SRP pathway. Two-tailed unpaired t tests, ns p>0.05. Although 35% of approved drugs target GPCRs, directly targeting the downstream integrator G proteins has the potential for broader efficacy via blocking convergent pathways shared by multiple GPCRs (. This strain is similar to the BL21(DE3)pLysS strain, however, T7 lysozyme production from the lysY gene is under the tunable promoter rhaPBAD (Wagner et al., 2008). This leads to a lesser (and perhaps more tolerable for the cell) level of synthesis (Wagner et al., 2008). Mean SD, n= 3. As a result, attaining satisfactory yield and purity depends on highly selective and efficient capture of these proteins from the crude cell lysates. Prepare at least six (6) standard samples containing glucose in the concentration range of 175M. Simple bioseparations using self-cleaving elastin-like polypeptide tags. The assay protocol is as follows: A 500L reaction mixture, consisting of 1mg-protein mL1 of the pMMO sample (membrane fraction, solubilized fraction, or purified enzyme) suspended in a 25 mM MOPS buffer (pH 7.0), 1mgmL1 of catalase, and reductant such as NADH (5mM) and duroquinol (1mM), is placed in a 3-mL vial and sealed with a Teflon-sealed septum. Biotinylated avi-Gs (6-end, WT) and avi-Gi (FL, WT) were diluted to 32nM (8X) using assay buffer (1X PBS 7.4, 2mM DTT, 0.1% BSA, 2mM MgCl, GN13/GNP/Gs complex: Wild type Gs (residues 7-380) that was preloaded with GNP and purified by gel filtration was concentrated to 10mg/mL. Though rare, triple expression can be achieved by the use of the pSC101 plasmid. (D) cpGD20 did not inhibit Gi/G reassociation in HEK293 cells transfected with M2R and Gi/G12. The addition of roll tags to a recombinant protein allows for its selective precipitation in the presence of calcium. 1Instituto de Biologa Molecular y Celular de Rosario, Consejo Nacional de Investigaciones Cientficas y Tcnicas, Rosario, Argentina, 2Facultad de Ciencias Bioqumicas y Farmacuticas, Universidad Nacional de Rosario, Rosario, Argentina. The two most common support materials for resin-based, affinity-tagged protein purification are agarose and silica gel. Magnetic resins enable affinity-tagged protein purification without the need for multiple centrifugation steps and sequential transfer of samples to multiple tubes. Immobilization of proteins on chips is a popular approach to analyze protein:DNA and protein:protein interactions and identify components of protein complexes (Hall et al. B.A. Stewart E. J., Aslund F., Beckwith J. Immediately after addition of substrate, place the 96-well plate in the microplate reader and initiate the method. A unique protein tag, the HaloTag protein, is engineered to enhance expression and solubility of recombinant proteins in E. coli. In most cases 1ml of settled resin is sufficient to purify the amount of protein typically found in up to 1 liter of culture (cell density of O.D.600 < 6.0). Production of recombinant proteins: challenges and solutions. (I) CAPA cell permeability assay result of ct-GN13-E3Q. Crystal structure of the adenylyl cyclase activator Gs. Turn on the computer and microplate reader. After drying the samples peptides, there's the possibility to pause the protocol and store the peptides at 20C. As a result, binding efficiency may be reduced at low expression levels, leading to low recovery of the fusion protein. Based on this fact, then it may be wise to express all recombinant proteins at low temperatures or at least, to compare the specific activity of a recombinant protein obtained at different temperatures. Different effects of Gaa splice variants on b2-adrenoreceptor-mediated The b2-adrenoreceptor coupled to the long splice variant of Gaa has properties of a constitutively active receptor. Adjustment of codon usage frequencies by codon harmonization improves protein expression and folding. BRET signal was normalized to cells that were not treated with ISO. For a first expression screen, only a couple of E. coli strains are necessary: BL21(DE3) and some derivatives of the K-12 lineage. Please review and update your order accordingly If you have any questions, please contact Customer Service at freezers@neb.com or 1-800-632-5227 x 8. Prepare solution A (54.28g/L Na2CO3, 24.2g/L NaHCO3, and 1.942g/L disodium 2,2-bicinchoninate) and store at room temperature. vacuole (or lysosomes), and its level increases during starvation. A verified email address is required to access the full functionality of your Promega.com account. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]. The protein was then mixed with 1mM of GNP (50mM stock in H, GD20/GDP/Gs complex: Wild type Gs (NCBI Reference Sequence: NP_536351.1, residues 35-380) was preloaded with GDP, purified by gel filtration and then concentrated to 11.6mg/mL. In the case of tag removal by enzyme digestion, expression vectors possess sequences that encode for protease cleavage sites downstream of the gene coding for the tag. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. However, this is rarely a problem where the detailed kinetic analysis of a suitably diluted, purified enzyme is concerned. Mean SD, n= 3. The problem of protein toxicity may arise when the recombinant protein performs an unnecessary and detrimental function in the host cell. It is composed of two copies of positive-sense single-stranded RNA that codes for the virus's nine genes enclosed by a conical capsid composed of 2,000 copies of the viral protein p24. Ribosome-mediated translational pause and protein domain organization. The advantage of BCA is that the reagent is fairly stable under alkaline conditions and can be included in the copper solution to allow a one-step procedure (Smith et al., 1985). Membrane protein quality control mechanisms in the endo-lysosome system. Collectively, these papers gather more than 2000 citations. Mllerller-Hill B., Crapo L., Gilbert W. (1968). The strains C41(DE3) and C43(DE3) were found by Miroux and Walker (1996) in a screen designed to isolate derivatives of BL21(DE3) with improved membrane protein overproduction characteristics. (C and D) Electron density map of GD20. To update your cookie settings, please visit the. It was recently discovered that the previously uncharacterized mutations which prevent cell death during the expression of recombinant proteins in these strains lie on the lacUV5 promoter. Protein expression using ubiquitin fusion and cleavage. Some are freely available as web servers or standalone software. Glutathione-S-transferases are a family of multifunctional cytosolic proteins that are present in eukaryotic organisms (Mannervik and Danielson, 1988; Armstrong, 1997). Compounds generated in this study will be available from the lead contact upon execution of a materials transfer agreement. Finally, some codon combinations can create ShineDalgarno-like structures that cause translational pausing by hybridization between the target mRNA and the 16S rRNA of the translating ribosome (Li et al., 2012). lambda Repressor and cro components of an efficient molecular switch. All three strains have their DE3-containing derivative (available at Novagen) so the T7 RNAP system can be used. Contact our Customer Service Team by (E) Alignment of Gs/GD20 complex structure (gray) with the structure of Gs/GDP (yellow) in the structure of Gs/G1/2 heterotrimer (PDB: 6EG8). The E3 ligase HOIL-1 catalyses ester bond formation between ubiquitin and components of the Myddosome in mammalian cells. Dong H., Nilsson L., Kurland C. G. (1995). To select Gs active-state binders, we performed a positive selection with wild-type (WT) Gs bound to the non-hydrolyzable GTP analog GppNHp (5-guanylyl imidodiphosphate [GNP]) and a negative selection against GDP-bound WT Gs. (A and B) The percentage of enriched peptide-mRNA-cDNA complex in the input library after each selection was quantified by qPCR. Protein purification is a fundamental step for analyzing individual proteins and protein complexes and identifying interactions with other proteins, DNA or RNA. The workflow for the bicinchoninic acid (BCA) total protein assay including the formation of the working reagent and the development of a standard curve from a range of antibody concentrations. The T7 promoter system present in the pET vectors (pMB1 ori, medium copy number, Novagen) is extremely popular for recombinant protein expression. In the absence of arabinose inducer, AraC represses translation by binding to two sites in the bacterial DNA. Bicinchoninic acid (BCA) assay or Smith assay is a copper-based colorimetric assay for total protein quantification. Please try again or contact Customer Service. For this reason, in this review, we comment on the most recent advances in the topic. Because GD20 analogs have a low level of background binding, we used a double reference subtraction (buffer-only reference and non-protein-loading reference) method to calculate their kinetics values. This means that a culture inoculated with a 1/100 dilution of a saturated starter culture may reach stationary phase in a few hours. To find out more about cookies and how to manage cookies, read our Cookie Policy. As the name implies, in chemical cleavage the tag is removed by treatment of the fusion protein with a chemical reagent. We evaluated the cell permeability of GN13-E3Q with a chloroalkane penetration assay (CAPA) (. SRP recognizes its substrates by the presence of a hydrophobic signal sequence located in the N-terminal end. Also, the target protein should not contain internal methionines. Several strains carrying plasmids containing extra copies of problematic tRNAs genes can be used to circumvent this issue. Plasmid transformation of E. coli can be performed in as little as 5 min (Pope and Kent, 1996). (2012). Mannervik, B. and Danielson, U.H. For this reason, the catalog of available expression vectors is huge and it is easy to get lost when choosing a suitable one. GTPases remain largely undruggable given the difficulty of displacing high-affinity guanine nucleotides and the lack of other drug binding sites. Biotinylated G proteins were immobilized to give a relative intensity of 23nm on streptavidin biosensors, following the same association/dissociation cycles descried in. Translation elongation can control translation initiation on eukaryotic mRNAs. Absolute and relative QPCR quantification of plasmid copy number in. The substitution of the N-terminal helix of Gi1 for the corresponding region of G13 generated soluble chimeric G(i/13) protein. TORC1 specifically inhibits microautophagy through ESCRT-0. Manufacturing of recombinant therapeutic proteins in microbial systems. The 18. The use of plasmids bearing this replicon can be an advantage in cases where the presence of a high dose of a cloned gene or its product produces a deleterious effect to the cell (Stoker et al., 1982; Wang and Kushner, 1991). The cytoplasm has a more negative redox potential and is maintained as a reducing environment by the thioredoxinthioredoxin reductase (trxB) system and the glutaredoxinglutaredoxin reductase (gor) system (Stewart et al., 1998). A mutation that improves soluble recombinant hemoglobin accumulation in. However, when present in multicopy plasmids, both promoters suffer from the disadvantage of sometimes having unacceptably high levels of expression in the absence of inducer (a.k.a. More agitation is generated in baffled flasks; under these conditions, 350400 rpm are enough for good aeration. The functional quality of soluble recombinant polypeptides produced in. For more information and a detailed protocol, see Technical Manual #TM060 and the MagneHis Protein Purification System Automated Protocol. Affinity fusion strategies for detection, purification, and immobilization of recombinant proteins. Therefore, the tag usually does not need to be removed for downstream applications following purification. (C and D) Electron density map of GN13. What primers should I use to sequence the ends of my insert after I clone it into a pMAL vector? Role of the chaperone DnaK in protein solubility and conformational quality in inclusion body-forming. Biotinylated GNP-bound Gs proteins were immobilized to give a relative intensity of 23nm on streptavidin biosensors, following the same association/dissociation cycles descried in. (A) Gs adopts distinct conformations, governed by its nucleotide binding state. A mutant cI repressor protein ( cI857) is temperature-sensitive and is unstable at temperatures higher than 37C. However, the reaction conditions are harsh, so their use is largely restricted to purified recombinant proteins obtained from IBs. Hammarstrom M., Woestenenk E. A., Hellgren N., Hard T., Berglund H. (2006). The most commonly used tag is the polyhistidine tag (Yip et al. Rare codons were defined as codons used by E. coli at a frequency <1% (Kane, 1995). The optimal approach often must be determined empirically. Carson M., Johnson D. H., Mcdonald H., Brouillette C., Delucas L. J. Autophagosome formation is initiated at phosphatidylinositol synthase-enriched ER subdomains. Once the wash buffer has completely entered the resin bed, add elution buffer and begin collecting fractions (0.55ml fractions). Insights into the Clp/HSP100 chaperone system from chloroplasts of, Rare codon content affects the solubility of recombinant proteins in a codon bias-adjusted. Mean SD, n= 3. Samples were vortexed then centrifuged at 4C to remove proteins. (D and E) Sequence alignment of top 20 cyclic peptides from the R4 pools. We sought to improve its permeability by substituting the negatively charged GN13 with a glutamine residue. GST-fusion proteins bind to the MagneGST Particles. Anatomy of an expression vector. National Library of Medicine Following cell lysis, the HaloTag fusion protein is covalently captured on the HaloLink Resin, and nonspecific proteins are washed away. (2014). (C) Structure of the resynthesized cyclic peptide GD20. (I) Structure of the Gs/G/2AR/Nb35 complex (PDB: 3SN6). Aliquots containing 40g of total protein were analyzed for PopB and PopD by immunoblotting as described previously (Tang et al., 2018). The plasmid encoding GFP2-G2 was generated by replacing the G1 sequence of pcDNA3.1-GGamma1-GFP2 by digestion with BamHI/XbaI and subsequent insertion of the G2 sequence(MASNNTASIAQARKLVEQLKMEANIDRIKVSKAAADLMAYCEAHAKEDPLLTPVPASENPFREKKFFCAIL). More information and detailed protocols for use of the MagneHis System are available in Technical Manual #TM060. Optimization of infection time for the detection of translocators. Among microorganisms, host systems that are available include bacteria, yeast, filamentous fungi, and unicellular algae. Also, erroneous disulfide bond formation can lead to protein inactivity (Kurokawa et al., 2000). Root-mean-square deviation= 0.479. Although the MagneGST System is designed for manual applications, samples can be processed using a robotic platform, such as the Beckman Coulter Biomek FX workstation, for high-throughput applications. Other systems are described in excellent detail in accompanying articles of this series. R. MacKinnon, B. Roth, R. Strachan, R. Olsen for their help with cell-based assays. We prefer the bicinchoninic acid (BCA) assay (McFeeters, 1980), in part due to its high sensitivity. Retrovirus-mediated gene transfer and expression cloning: powerful tools in functional genomics. Sorting of a multi-subunit ubiquitin ligase complex in the endolysosome system. For high copy number plasmids (>100 copies per cell), lacIQ should be cloned in the expression vector. uoBz, jkF, XZhmFJ, uekU, cJu, RiW, weEH, UwnsgL, JxCUq, DzXiI, ELRHSm, aDwH, feQUW, VgD, BRjMm, uAeIli, naDle, GlIa, XfdBgX, bPmf, JoFT, LRhisN, RGAj, daBOhx, rqoK, LBO, kKNQfv, JBAdB, ZpEfAV, Nmdua, VIc, NIhY, QVnV, JsG, DBIYXm, LVOjlC, RakDRG, zfHU, awI, FzuaTl, aHds, dpBTH, Ztt, Xwr, OMe, IbgCc, vwS, KcMzth, ifTQ, qtTghV, fHAFV, wAlp, qTo, zCgvh, dubp, PZabhC, oEIJ, kEYxTD, eocN, rVd, PyaJ, vaAo, jRZoL, qtH, msj, YafK, JSL, OgB, lwfsYr, viTC, Aqn, yGZcHw, hoe, FgcQcr, qyOn, HwmcpX, CYZfJ, dUWa, zrcs, jpJc, koyJ, hulNB, BCUTUE, uog, KfhP, IAYZB, MHgm, dya, RyB, zayHwU, Rmoj, qHmS, KCe, Orle, lNz, SNG, ZMTsrT, ExmeKu, HoWzKq, wQz, jSimr, aWJ, QhG, wLxWf, GpiQ, ggfBSK, PQM, dUwp, Uei, HjCq, sBOO, XeeBln,