To overexpress and purify the His-TEV protease from E. coli bacteria. Cleanrooms or other controlled environments used for research or manufacturing need specialized products. Standard deviations for the kcat/KM values were calculated as described (Boross et al., 1999). Webnon-trypsin protease standard curve and unknown sample) are low (e.g., < 0.3) pH and other buffer conditions were not optimal for the protease being tested Repeat assay using buffer conditions that are optimal for the protease being tested and used to make standard curve Protease being tested digests the casein substrate more slowly or On the other hand, no degradation of the F217K mutant was evident even after 8 h under the same conditions. So much has changed during this unprecedented time, except your ability to count on Avantor. stream MeSH Kinetic analysis of the TEV protease mutant S219P. (. The amino acid residues corresponding to the P2 and P1 sites were targeted for mutagenesis because these were the only positions that matched the canonical (consensus) TEV protease recognition site; suboptimal residues were present in the other positions. Compatible with the following protease inhibitors: aprotinin, benzamidine, leupeptin, pepstatin, PMSF. total units of the original product. The amino acid composition of the peptides was determined with a Beckman 6300 amino acid analyzer. Allosteric activation of autoproteolysis. The optimal temperature for cleavage is 30C; however, the enzyme can be used at temperatures as low as 4C. In contrast to a previous study (Parks et al., 1995), we found that the rate of autoinactivation is proportional to the concentration of TEV protease. Contact VWR Custom Manufacturing Services at 1.800.932.5000 or VWRCustom@vwr.com. TEV protease cleaves the amino acid sequence ENLYFQG/S between QG or QS with high specificity (Parks et al., 1994). Clipboard, Search History, and several other advanced features are temporarily unavailable. (, Wagner,C.R., Bergstrom,C.P., Koning,K.R. Avantor can help you plan, design, and configure a custom laboratory environment that meets all your needs. For more information, call 1.888.793.2300 or email us at services@avantorsciences.com. WebN-Hydroxylating monooxygenases (NMOs) are involved in siderophore biosynthesis. The peptide was hydrolyzed by the wild-type and mutant enzymes between Gln and Ser, as expected. Durable, low-linting, & available in white or blue. The autocatalytic site of NIa at S2256 has been mutated to an N for improved stability of the protease. Update to Avantors response to the coronavirus (COVID-19) pandemic. WebIt is recommended that the cleavage for each fusion protein be optimized by varying the amount of recombinant viral TEV protease, reaction time, or incubation temperature. Xhw-6=:Wh.gg'hh|a,e'@JDug{7 `az+}r}86Cj79cq]?|7XDY7 Nilsson,J., Stahl,S., Lundeberg,J., Uhlen,M. Avantor carries a broad range of trusted products to help you meet the needs of your lab applications and protocols. During the course of this work, we made some interesting observations pertaining to the mechanism of autoinactivation. WebThe Center will: 1) plan, develop and conduct research studies on the molecular structure and interactions of biological macromolecules and therapeutics pertinent to human health, with Don't miss out, order yours today! Aliquots were removed at regular intervals over 24 h and the extent of self-processing was monitored by SDSPAGE. However, the sequence specificity of TEV protease is not absolutely stringent because all but one of the natural processing sites in the TEV polyprotein deviate from the consensus sequence and TEV protease can cleave many engineered sites that do not exactly match the canonical target site (Dougherty et al., 1988,1989). and Waugh,D.S. Epub 2011 Aug 31. f?3-]T2j),l0/%b The rate of autoinactivation (V; nmol/dm3/min) at various protease concentrations [E] was calculated from these data under conditions where less than 20% of the protease was cleaved. Methods Mol Biol. The column was washed with 15 column volumes of buffer A containing 25 mM imidazole, after which the HisTEVArg protease was eluted with buffer A containing 200 mM imidazole. Avantor is a vertically integrated, global supplier of discovery-to-delivery solutions for We found alternative products that can save you up to per item-unit. and Lake,M. A systematic analysis of the P1 specificity of TEV protease revealed that Glu (which is very similar to Asp), the -branched hydrophobic residues (Leu, Ile and Val), and especially Pro are the most unfavorable residues in this position (R.Kapust et al., unpublished observations). The canonical TEV recognition site is displayed below the potyvirus sequences and aligned with the internal cleavage site in TEV protease; amino acid residues that are least tolerant of substitution (Dougherty et al., 1989) are underlined. TEV Protease has a 7xHis-tag for easy removal from a reaction using nickel affinity resins and has been engineered to improve thermal stability and decrease autolysis. VAI's cellulose free, low particlate and chemical resistant cleanroom paper. Appl Environ Microbiol. 2023;2551:41-51. doi: 10.1007/978-1-0716-2597-2_4. and Nygren,P.A. This sample was then applied at a flow rate of 3 ml/min to a 2.660 cm (320 ml) Sephacryl S-100 HR column equilibrated in buffer F: 20 mM bicine (pH 8.5), 2 mM EDTA, 2 mM 2-mercaptoethanol, 500 mM NH4Cl. New Avantor J.T.Baker premium conductive and non-conductive robotic tips deliver superior quality and reliable performance for results you can trust. Life Science Research Solutions, Products, and Resources, Controlled Environment and Cleanroom Solutions, SureSTART Autosampler Vials and Wellplates, Rheo Flexibles: Single Use Containment & Transfer Technology, Thermo Scientific Chromatography Columns and Consumables, Liquid Chromatography - Sample Preparation, Biopharma production chemicals & excipients, Laboratory Personal Protection Equipment (PPE) and Workwear Products, Laboratory Facilities Maintenance, Protection, and Hygiene Supplies, Building a Safe Work Environment with VWR, Aerospace & Automotive Industry Cleanroom Products, Pharmaceuticals, Biotechnology and Nutraceuticals, Micronova - Contamination Control Made Simpler, Quality Products from Sheldon Manufacturing, Agilent Chemistries and Supplies Portfolio, PerkinElmer - Innovating for a Healthier World, Sartorius laboratory instruments, consumables and services, HALO Chromatography Columns and Consumables+-, VWR Returnable Container Advantage Program (ReCAP), The Avantor Value as a Medical Lab Partner, Reference materials Laboratory Products, Specialized Solutions for Cutting Applications, Contact Your Laboratory Furniture Specialist, Removal of MBP and His affinity purification tags from fusion proteins, Optimal activity and stability for up to 24 months, Active in a wide range of buffers; optimal activity between pH 6.0 and 9.0, High substrate specificity with no non-specific proteolysis, TEV Protease contains a His-Tag for removal from a reaction using NEBExpress Ni Resin (NEB# S1428). The S219E and S219V proteases were converted to the truncated form at a rate that is ~10-fold slower than the S219D mutant (compare S219E and S219V after 24 h with S219D after 2 h) or ~100-fold slower than the wild-type protease. Avantor can help maintain cleanroom standards with protective apparel and environmental monitoring equipment. and Brady,K.D. We continue to set science in motion to create a better world by providing you with the right solutions to keep moving forward. 299321. Weve built our reputation on consistent, comprehensive mastery of day-to-day operations, allowing lab, clinical, and production environments to focus their high-value resources on core scientific priorities. 1 x glycerol stock of HisTEV protease in BL21(DE3)-RIL cells (AddGene 8827 pRK793) 1 x LB + amp. Systematic studies have implicated E, Y, Q and, to a lesser extent, G/S, as important specificity determinants (Dougherty et al., 1988,1989). The nuclear inclusion protease from tobacco etch virus (TEV) is one of the best-characterized enzymes of this type. The fraction of protease that was cleaved as a function of time at each concentration was estimated by laser-scanning densitometry of the stained gels, using a Personal Densitometer (Molecular Dynamics). Thus, none of the mutations at the S219 position adversely affect the stability of TEV protease. The reaction mixtures were incubated at 30C for 30 min, then stopped by the addition of 160 l of 4.5 M guanidine hydrochloride (Gd-HCl) containing 1% trifluoroacetic acid (TFA). Because the HisTEVArg proteases become highly concentrated during purification, a significant amount of the truncated protease sometimes formed during the first two chromatographic steps, particularly in the case of wildtype HisTEVArg. (, Forsberg,G., Brobjer,M., Holmgren,E., Bergdahl,K., Persson,P., Gautvik,K.M. This discrepancy is probably due to the fact that the highest concentration examined by Parks et al. Accessibility WebTEV protease has activity in the pH range of 6-9. Our product offerings can help with biomedical research, forensic work, and clinical diagnostics. ^:tTM8yfI@] H" f^[JaI]:JKZ,Uy/um46x'pI-]Cg{/Yl|oV_d'CNM.NvrL)A_X=@XD^\K)O{qbn+2fA#p#FZUIV,\Y#%y.$+Q>B_*|P#G^1buCyfFjZKA}WTrRGg?5}i;tlH$B&^9lk+L&WL`XA~~T)_"~Mdtm:v6`:j5}Aq\G3U3aIf:t0$0dU@. An enzymatically biotinylated, catalytically inactive TEV protease mutant (D81N) was incubated alone or in combination with an equimolar amount (67 M) of catalytically active, wild-type HisTEVArg protease in reaction buffer for 1 h at 30C. Abbreviations: Ppvna, plum pox virus (isolate NAT); Ppvd, plum pox virus (strain D); Ppvra, plum pox virus (strain Rankovic); Ppvea, plum pox virus (strain El Amar); Pvyn, potato virus Y (strain N); Pvyhu, potato virus Y (strain Hungarian); Pemvc, pepper mottle virus (California isolate); Tev, tobacco etch virus; Tvmv, tobacco vein mottling virus; Psbmv, pea seed-borne mosaic virus; Wmv2, watermelon mosaic virus 2. As shown in Figure 3, autoproteolysis of wild-type HisTEVArg protease was stimulated by the addition of catalytically inactive protease. We thank Karen Routzahn for constructing pKM607 and pKM773, Suzanne Specht for assistance with peptide synthesis and amino acid analysis and Anne Arthur for expert editorial advice. 4H@R{t|zGU*e{${|Em. &OJF#wcu"h (.7T9r?'E^#y6(y& = z~nh W v b?\r]3N1 B: 9_FU| W>!GACr 4NLa{ Strickland KA, Holland AA, Trudeau A, Szlamkowicz I, Beazley MJ, Anagnostopoulos VA, Graham DE, Caranto JD. Avantor supports end-to-end fluid management solutions including peristaltic pumps and aseptic fluid transfer solutions that are reliable and customer-centric, helping bioprocessing manufacturers meet their research and production goals. %PDF-1.4 (, Dougherty,W.G., Cary,S.M. Exceptional skid resistance, both wet and dry, for dirty jobs and controlled environments. This chapter describes a simple method for overproducing a soluble form of the tobacco etch virus (TEV) protease in Escherichia coli and purifying it to homogeneity so that it may be used as a reagent for removing affinity tags from recombinant proteins by site-specific endoproteolysis. Learn more and request a sample! Moreover, the progressive loss of activity during storage of the enzyme is a considerable nuisance. The mutations were introduced into pRK683 by overlap extension PCR (Ho et al., 1989), using a complementary pair of internal primers containing the desired nucleotide substitutions in concert with PE-29 and PE-30 (5-GCA AGG CGA TTA AGT TGG GTA ACG C-3), a pair of external primers that flank the TEV protease ORF. To investigate this possibility, we incubated a fixed amount of the wild-type HisTEVArg protease (~17 M) with increasing concentrations of the catalytically inactive BAPTEV(D81N)His protein and monitored autoinactivation as a function of time. The fractions containing biotinylated BAPTEV(D81N)His protease were pooled and concentrated to 22 ml. Federal government websites often end in .gov or .mil. An official website of the United States government. (, Gorbalenya,A.E., Donchenko,A.P., Blinov,V.M. It has been suggested that autoinactivation of TEV protease may play a role in the physiology of viral infection (Parks et al., 1995; Kim et al., 1996). On the other hand, the closely related tobacco vein mottling virus protease evidently does not undergo autoinactivation in vitro (Hwang et al., 2000), so this does not appear to be a universal property of potyviral proteases. For maximum convenience and value, columns and buffers are also available separately. The latter tag is a substrate for enzymatic biotinylation in E.coli (Schatz, 1993). Adding products to your cart without being signed in will result in a loss of your cart when you do sign in or leave the site. Bethesda, MD 20894, Web Policies Solid ammonium sulfate was added to the supernatant to 35% saturation, after which the solution was clarified again by centrifugation as above. The structure of Geobacillus stearothermophilus purine nucleoside phosphorylase, an enzyme of biocatalytic interest, is reported and was found to include the presence of an N-terminal tag in the active site of each subunit that belongs to the other subunit in each dimer. The nucleotide sequence of the insert was verified experimentally. One of the main uses of this protein is for removing affinity tags from purified recombinant fusion proteins. The reason for the use of TEV protease as a biochemical tool is its high sequence specificity. This specificity allows for the controlled cleavage of proteins when the preference sequence is inserted into flexible loops. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. (, Laco,G.S., Fitzgerald,M.C., Morris,G.M., Olson,A.J., Kent,S.B. The six-membered ring of the pipecolic acid moiety is solvent exposed and is stabilized by the phenyl ring of F117 and the hydrophobic side chains of L97, L119, and L196 (Fig. In addition to vwr.com, VWR, part of Avantor offers a state-of-the-art technology solutions portfolio. Add 5 l of TEV Protease Reaction Buffer (10X) to make a 50 l total reaction volume. Epub 2022 Sep 26. In this regard, it is intriguing to note that autoinactivation also occurs in some related proteases, including turnip mosaic virus protease (Kim et al., 1996) and hepatitis A virus 3C protease (Gauss-Muller et al., 1991), albeit at different locations. The proteases that are most often used are activated blood coagulation factor X (factor Xa), enteropeptidase (enterokinase) and -thrombin, yet the literature is replete with reports of fusion proteins that were cleaved by these proteases at locations other than the designed site (Forsberg et al., 1991,1992; He et al., 1993; Wagner et al., 1996; Stevens, 2000). In the original Tango design, the TEV protease and actuator module (tetracycline transactivator proteintTa) with its TEV protease cleavage site were fused to an adaptor beta-arrestin and the C-terminus of a GPCR, respectively. In an effort to create a more stable but equally active form of TEV protease, we constructed several mutants with single aminoacyl substitutions adjacent to the internal cleavage site and investigated their impact on the stability and catalytic activity of the enzyme. The concentration can be calculated using The His-tagged TEV protease can be purified in two steps using immobilized metal affinity chromatography (IMAC) followed by gel filtration. (, Oxford University Press is a department of the University of Oxford. These gloves offer the perfect combination of improved elongation with outstanding protection from chemotherapy drugs, and an FDA-approved low dermatitis potential claim. Optimal activity achieved in 0.2M NaCl; however, the enzyme retains some activity in up to 2M NaCl. WebAbstract. The fractions containing biotinylated BAPTEV(D81N)His protease were pooled, concentrated to 2 mg/ml, mixed with an equal volume of glycerol, flash-frozen in liquid nitrogen and stored at 80C. Assortment of popular, everyday Nalgene labware in a convenient pack. Direct assay for intermolecular autoinactivation of TEV protease. To purify the catalytically inactive BAPTEV(D81N)His protease, 4 g of cell paste was thawed and resuspended in 40 ml of buffer D: 25 mM HEPES (pH 8.0), 75 mM NaCl, 10% (w/v) -sorbitol. However, the larger product PTTENLYFQ, which had approximately four times the molar extinction coefficient of the smaller product, comigrated with the substrate during HPLC, while the other product partially comigrated with the DTT peak in the chromatogram, making it difficult to determine precise kinetic parameters. Would you like email updates of new search results? WebProtein information: TEVP: Extinction coefficient: 32,290 M-1cm-1, MW: 28.6 kD GFP-POI: Extinction coefficient: 63,300 M-1cm-1, MW: 65.9 kD GFP: Extinction coefficient: The nucleotide sequence of the insert in each vector was confirmed experimentally. Monarch Nucleic Acid Purification Kits are optimized for maximum performance and minimal environmental impact. The addition of two extra Arg residues had a beneficial effect on the solubility of these substrates. E-mail: waughd@ncifcrf.gov. Surprisingly, as shown in Figure 2, the catalytically active HisTEVArg protease was unable to convert the catalytically inactive BAPTEV(D81N)His protease into the truncated form. An Efficient Method of Expression and Purification of Amyloid-Beta (A. Optimal incubation times and enzyme concentrations must be determined empirically for a particular substrate. Methods Mol Biol. How is savings calculated?We Q)1> mL/Xy=18iOO PK ! To ascertain whether either of these mutations interferes with the catalytic activity of TEV protease, we determined the kinetic parameters Km and kcat for processing of a canonical peptide substrate by wild-type and mutant forms of HisTEVArg. Consequently, if any of the inactive (biotinylated) protease was cleaved by the active HisTEVArg enzyme, then we would be able to detect the truncated product with a streptavidin-horseradish peroxidase probe in a western blot experiment. These seemingly paradoxical observations could be reconciled if the mechanism of autoinactivation involves an allosteric interaction between TEV protease molecules that triggers intramolecular proteolysis. The intact protease and its degradation product were separated by SDSPAGE and visualized by staining with GelCode Blue. WebAdd TEV protease at a protease to target protein ratio of 1:100 (w/w) or 10,000 unit (1 mg) TEV protease to 100 mg of target protein. Avantor, a Fortune 500 company, is a leading global provider of mission-critical products and services to customers in the biopharma, healthcare, education & government, and advanced technologies & applied materials industries. High-throughput protein purification and quality assessment for crystallization. 2007 Jan;51(1):102-9. doi: 10.1016/j.pep.2006.07.003. agar plate 1 x 100 mL LB + amp. TEV protease has (NIa) protease catalytic domain which corresponds to a molecular weight of 27 kDa. It is unique with high specificity and is active at low temperature. The tobacco etch virus (TEV) protease is a useful tool for the removal of fusion tags from recombinant proteins. The products you use, the products you need, the suppliers you trust for chromatography. Interacts with virions and aphid 2011 Sep;55(1):12-28. doi: 10.1016/j.ymeth.2011.07.010. TEV protease can be added directly to the target protein. To separate the truncated material from the full-length protease, the dialyzed sample was applied to a column (1.610 cm) of Merck Fractogel EMD-COO (2040 M) resin, equilibrated with buffer C, at a flow rate of 2 ml/min. The pellets were resuspended in ice-cold TEV protease reaction buffer: 50 mM TrisHCl (pH 8.0), 0.5 mM EDTA, 1% glycerol, 1 mM dithiothreitol (DTT). However, a slight amount of the truncated protease appeared after overnight incubation (24 h) of the F217K mutant. Autodigestion of TEV protease occurs between Met218 and Ser219 (Parks et al., 1995), causing a nearly 10-fold reduction in proteolytic activity (Table I). Bookshelf These goggles are comfortable and have good peripheral vision. N _rels/.rels ( j0@QN/c[ILj]aGzsFu]U ^[x 1xpf#I)Y*Di")c$qU~31jH[{=E~ Before Add 5 l of TEV Protease Reaction Buffer (10X) to make a 50 l total reaction volume. They are also among the least favorable substitutions at these positions in a canonical TEV protease recognition site (Dougherty et al., 1988,1989). An aliquot was injected onto a Nova-Pak C18 reversed-phase chromatography column (3.9150 mm, Waters Associates) using an automatic injector. x]YsGyOI8kfVWUfugz KRj~-W'_kJv|Sz}SMtA9vP*cYg "?.UP@LdBsR[W'y:f Je{{]$0RT@dtRpu`h^W?NbbG=trXTF**}iQrUJlI;3WIh_K.mLwlErhyCtKJ-Yo0=OH! The PCR products were digested with SacI and BamHI, and then ligated between the unique SacI and BamHI sites in pMal-C2 to create the corresponding Escherichia coli maltose-binding protein (MBP) fusion vectors. This general strategy had been used successfully in the past to inhibit the autoinactivation of other proteases (Rose et al., 1993; Dang et al., 1996; Laco et al., 1997; Tomasselli et al., 1998; Louis et al., 1999; Pray et al., 1999). Although affinity tags are highly effective tools for the expression and purification of recombinant proteins, they generally need to be removed prior to structural and functional studies. The VWR Traceable Logger-Trac Temperature Datalogger is perfect for monitoring material during storage, handling, and transportation. Autodigestion of wild-type (WT) and mutant TEV protease catalytic domains. It is becoming increasingly evident that certain viral proteases have more stringent sequence specificity (Babe and Craik, 1997). The cells were lysed with an APV Gaulin G1000 homogenizer at 10 000 psi, after which the insoluble debris was pelleted by centrifugation at 15 000 g for 30 min. A unimolecular reaction mechanism might explain the relatively facile autolysis of TEV protease at a site that bears little similarity to the canonical recognition sequence (Parks et al., 1995). The column was washed with seven volumes of buffer E, after which the biotinylated BAPTEV(D81N)His protease was eluted with buffer E containing 2 mM biotin. Methods. The propensity of the mutant proteases to undergo autoinactivation was assessed by incubating them under reaction conditions typically used to digest fusion protein substrates. Careers. "?~_vEpEi;#(t$Y}wtG8,(-58]]"Aq2yFEMPo>-[D)yWAe8O_e|'qy 1wG<=qi5V_qk The supernatant was then adjusted to 65% saturation with solid ammonium sulfate and incubated on ice for 15 min. BL21/DE3 cells containing pRK810 were grown, induced and harvested as described above for the HisTEVArg proteases, except that the medium was supplemented with 50 M biotin. An alternative interpretation of our results is that autolysis of TEV protease is an intramolecular event that is stimulated by an allosteric interaction between protease molecules, reminiscent of the manner in which E.coli RecA stimulates autolysis of the LexA repressor (Little, 1984). The supernatant was applied at a flow rate of 2 ml/min to a 1.65.0 cm (10 ml) Ni-NTA column that had been equilibrated with buffer D. The column was washed with three volumes of buffer D followed by seven volumes of buffer D containing 30 mM imidazole. Proc. WebThe protease can be used for the removal of affinity tags from fusion proteins. This serves as a warning that while tags enable simple and economic The protein concentrations were estimated on the basis of their absorbance at 280 nm (extinction coefficient = 32 410 M 1 cm 1 ), after which the proteases were All of the mutant TEV proteases exhibited a greatly reduced propensity for autoinactivation. Autoprocessing of the wild-type and mutant fusion proteins proceeded to completion in vivo to yield separate MBP and HisTEVArg domains. Autoproteolysis of TEV protease was monitored as a function of time, by SDSPAGE, at a series of protein concentrations ranging between 1 and 120 M (3.5 mg/ml), and the results were quantified by laser-scanning densitometry of the stained gels. The fusion protein subsequently cleaves itself in vivo to remove the MBP moiety, yielding a soluble TEV protease catalytic domain with an N-terminal polyhistidine tag. To investigate this possibility, we examined the concentration dependence of the autoinactivation reaction. Most enzymes are proteins, although a few (. 2022 Nov 10;13(1):6820. doi: 10.1038/s41467-022-34654-2. Conversely, the S219D mutant exhibits wild-type catalytic efficiency but still undergoes autoinactivation at an appreciable rate. Add 1 l of TEV Protease. In order of efficiency, S, A, M, Y, D, N, E, K or L may be effectively used in place of G. Several of the remaining aa may also vary, giving a final consensus sequence of ExxYF(M)Q(E)/G(S, A or others) where aa in parenthesis are alternatives and x is any aa. The HisTEVArg proteases were generated by intracellular autoprocessing of MBP fusion proteins as described (Kapust and Waugh, 1999), except that the temperature was reduced to 30C upon addition of isopropyl --thiogalactopyranoside (IPTG). Wild-type and mutant TEV proteases were diluted to an A280 value of ~0.05, in the presence of 20 mM sodium phosphate (pH 7.5), 5 mM DTT and various concentrations (02.8 M) of Gd-HCl. Therefore, we constructed and characterized HisTEVArg proteases containing these three mutations (S219E, S219V and S219P). These proteases adopt a trypsin-like fold but possess an unconventional catalytic triad in which cysteine replaces serine (Bazan and Fletterick, 1989; Gorbalenya et al., 1989). The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Some substrates may require extended incubation periods (up to three days at either 4C or 30C) to achieve complete cleavage. Our global footprint enables us to serve more than 225,000 customer locations and gives us extensive access to research laboratories and scientists in more than 180 countries. An equal concentration of bovine serum albumin failed to stimulate autolysis of wild-type HisTEVArg protease, and the addition of catalytically inactive protease neither accelerated nor inhibited the processing of a canonical fusion protein substrate by the wild-type protease (data not shown). Please enable it to take advantage of the complete set of features! WebThe His-tagged TEV protease can be purified in two steps using immobilized metal affinity chromatography (IMAC) followed by gel filtration. The 29 kDa HisTEVArg proteases and their 26 kDa degradation products were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) on 12% Trisglycine gels (Novex). and Dougherty,W.G. VWR/Anachemia continues to be the undisputed leader when it comes to supplying laboratories conducting mineral analyses around the world VWR is proud of our years of experience providing choice and excellent service to the Industrial market from Food & Beverage, Petrochemical, Environmental Testing, Waste Water, Cosmetics, Consumer Goods, Agriculture and more See how the unique Nalgene Rapid-Flow membrane support system provides the last line of defense against contamination. 4ze H word/_rels/document.xml.rels ( N0EHC=qByi7[({FZ,s[u'j,Xf,,l.% e:m&m'm6= }QQijCGY6f'=,YH_nzOo]Um \+@w7 $(L zn*!h`c"?+yc}1!uupcZPG HMau(pRp Initial velocities at six substrate concentrations were fitted to the MichaelisMenten equation. doi: 10.1371/journal.pone.0266488. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1 positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. HHS Vulnerability Disclosure, Help Equilibrium denaturation of wild-type (wt) and mutant TEV proteases. Incubate at 30C for 1 hour or at 4C overnight. The kcat of the S219V mutant was somewhat better than that of the wild-type protease, whereas the corresponding values for the S219E and S219P mutants were ~5080% that of wild-type HisTEVArg. doi: 10.15252/embj.2022111540. PK ! The values of Km for the S219E, S219V and S219P mutants were essentially the same as that of the wild-type protease (Table I). The full-length HisTEVArg protease was eluted with buffer C containing 500 mM NH4Cl. The protein was eluted from the column with three volumes of buffer B containing 1 M ammonium sulfate. Therefore, you need to exchange the buffer of the protein solution immediately after elution from the The latter mutant exhibited only a 2-fold reduction in kcat and the kinetic parameters determined for the S219V mutant were as good or better than those of the wild-type protease. and Craik,C.S. qaSt\Z..8Tly)]Ma@ .RLL@k0Za>:6i$?'U|X0>? The result indicates that the multiple mutations given both to improve the yield of TEV protease and to prevent self-inactivation completely solve the problems at once and even increase TEV activity further. We thank Dr. Seonhun Kwon for helpful discussion. There is 75 mM reduced If you don't see your country above, please visit our WebTEV Protease is a highly specific cysteine protease that recognizes the amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves between the Gln and Gly/Ser residues.Enzymes accelerate, or catalyze, chemical reactions, and they are known to catalyze more than 5,000 biochemical reaction types. P program (Fig. This behavior is inconsistent with a unimolecular reaction mechanism. Using this procedure, we obtained ~2.5 mg of pure HisTEVArg protease per gram of wet cell paste. The pure proteases were immediately mixed with an equal volume of glycerol, aliquoted, flash-frozen in liquid nitrogen and stored at 80C until further use. 2009;498:157-72. doi: 10.1007/978-1-59745-196-3_11. We have become experts in scientific operations, improving performance with sophisticated solutions and providing guidance on best practices. Quanta Biosciences and VWR are proud to fund a grant award for publication of research involving qPCR methodologies utilizing any Quanta Bioscience qPCR reagent in JoVE. Our latest RUO kit, the Luna SARS-CoV-2 RT-qPCR Multiplex Assay Kit, enables high throughput workflows for real-time detection of SARS-CoV-2 nucleic acid using hydrolysis probes. If the fusion protein sample contains >2 M urea, >0.5 M Guanidine hydrochloride, >50 mM imidazole, pH values below 6 or above 9, or cysteine protease inhibitors then it will be necessary to dialyze the fusion protein into TEV protease reaction buffer before TEV Protease cleavage. The 29 kDa protease and its 26 kDa degradation product were separated by SDSPAGE on 12% Trisglycine gradient gels. WebTEV protease is not stable in buffers containing a high concentration of imidazole. Enhancing the solubility of recombinant proteins in Escherichia coli by using hexahistidine-tagged maltose-binding protein as a fusion partner. @oE[{0ucfucdK_&]3kYc6LneU @X+X!h2ZmL2XV` a(j PwfHH~~Co\ocN\C[m:IGD)Z@k( BP%c9|m o-83x$XPQ=.uQDMTM',O2ZR7>!WjN` o&JaE #7[QF\ iiggPdK*~av^q;@iLwu|oHFpuD)Rn#*/)Q[\?>Q~1,eoibKhe~.^Ez'9d8*1z_k}}u.SL/v$)k0c?.mAK*6/H?To^C=mnsxxba\N.iY3d1391ko0QIQI;Kfn)J>OgT]M8qG>)A"dXx$gEG\/%+1w ^,GYA3p)FkWRjuyOuFQg7m? The HisTEVArg proteases (33 M, 1 mg/ml) were incubated in reaction buffer for various times ranging between 0 and 24 h, as indicated. For full access to this pdf, sign in to an existing account, or purchase an annual subscription. Sci. Kinetic parameters were determined by fitting the data obtained at less than 20% substrate hydrolysis to the MichaelisMenten equation by using the Fig. (1972) Experiments in Molecular Genetics. The protein concentrations were estimated on the basis of their absorbance at 280 nm (extinction coefficient = 32 410 M1 cm1), after which the proteases were diluted to the desired concentration (33 M) with reaction buffer and incubated at 30C. This sample was then applied to a 1.620 cm column of Ni-NTA-agarose (Qiagen) pre-equilibrated in buffer A at a flow rate of 2 ml/min. Thus, these kinetic experiments revealed that the F217K mutation has a deleterious effect on the catalytic activity of TEV protease, due to a Km effect, which may account for its greater degree of resistance to autoinactivation than the S219D mutant. Recombinant proteins are often fused to various peptide and protein partners to facilitate their detection and purification, increase their yield and enhance their solubility (Uhlen et al., 1992; Nygren et al., 1994; LaVallie and McCoy, 1995; Nilsson et al., 1997; Baneyx, 1999). Typical reaction conditions are as follows: Combine 15 g of substrate and H 2 O (if necessary) to make a 45 l total reaction volume. Try the improved Chemical Structure search through the new. (~8 M) corresponds to the lowest part of the curve in Figure 1, where the concentration dependence is far less pronounced. Alignment of amino acid sequences near the C-termini of potyvirus nuclear inclusion protease catalytic domains. Hexahistidine-tagged maltose-binding protein as a fusion partner for the production of soluble recombinant proteins in Escherichia coli. Kits are available for total RNA purification, plasmid miniprep, gel extraction, and DNA & RNA cleanup. (, Gauss-Muller,V., Jurgensen,D. Inhibition occurs in the presence of 5 mM Zn. (, Parks,T.D., Leuther,K.K., Howard,E.D., Johnston,S.A. and transmitted securely. The final substrate concentrations ranged between 0.03 and 1.4 mM; the actual range was selected on the basis of the approximate Km value for each protease. High-quality chemicals and services, customized to your product or manufacturing needs VWR enables the advancement of science by providing high-quality chemicals and services, customized to your product or manufacturing needs. As shown in Figure 5, the majority of the wild-type protease was converted to the truncated form after only 2 h at 30C. As expected, all three of these mutants proved to be far more resistant to autoinactivation than the S219D protease (Figure 5). Journal of Biotechnology 121, The eluted material was dialyzed against 20 mM bicine (pH 8.1), 20 mM NH4Cl, 1 mM EDTA, 20 mM 2-mercaptoethanol (buffer C) containing 10% (w/v) -sorbitol until the conductivity had dropped below that of 50 mM NH4Cl. Due to its high sequence specificity it is frequently used for the controlled cleavage of fusion proteins in vitro and in vivo. (, Pray,T.R., Nomura,A.M., Pennington,M.W. PMC Perhaps the scissile bond is contained within a relatively flexible segment of the protein that is positioned very close to the active site, effectively mimicking a highly concentrated substrate. and Choi,K.Y. Each time, ~17 ml of BAPTEV(D81N)His protease was applied at a flow rate of 1 ml/min to a 1.64.0 cm (7 ml) column containing UltraLink immobilized monomeric avidin (Pierce) that had been equilibrated in buffer E: 25 mM TrisHCl (pH 7.5), 25 mM NaCl, 10% (w/v) -sorbitol. sharing sensitive information, make sure youre on a federal o gHhAa]sN#~nnD)-_?pD[X5GBe,(r*,vP)^2shH9y] +Wpcg,.vuZKn( f+PyuE0_q/u!F BA o"&3`} The concentration of protease in the reactions was 125 nM. The seven-amino-acid recognition site for TEV protease is Glu-Asn-Leu-Tyr-Phe-Gln-Gly with cleavage occurring between Gln and Gly. The Km and kcat values for the wild-type and mutant HisTEVArg proteases are presented in Table I. <> The values of Km and kcat that were obtained for wild-type TEV protease are in excellent agreement with previous results (Parks et al., 1995), indicating that the addition of five arginine residues to the C-terminus of the catalytic domain does not interfere with its activity. Thus, the S219D mutant appears to be approximately 10 times more resistant to autoinactivation than the wild-type protease under these conditions. It may be used in biotechnology to cleave affinity tags from recombinant proteins, either co-translationally or, Custom Services for Cell & Gene Therapies, Good Manufacturing Practice (GMP) Antibody Services. Although both chemical and enzymatic methods have been used to cleave fusion proteins at designed sites (Nilsson et al., 1997; Southworth et al., 1999), only the natural proteolytic enzymes have the requisite specificity to be broadly useful reagents for this purpose. OVEREXPRESSION Materials. We use operational excellence to deliver solutions that enable research, testing, production, and commercialization across the globe. 7!Kxq?>^=o7d$[C?4O?Xc\dDq! WebTEV protease is a highly specific cystein protease belonging to the C4 peptidase family. and Parks,T.D. The autoinactivation-resistant mutants described here could be used to clarify what role, if any, autoinactivation plays in the regulation of TEV protease activity during viral replication. An amino acid substitution in the P2 position of the internal cleavage site (F217K) virtually eliminated autoinactivation under normal reaction conditions. 8600 Rockville Pike This site needs JavaScript to work properly. Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. If, as we have suggested, autoinactivation is triggered by an allosteric interaction between TEV protease molecules, then a regulatory role seems plausible. WebTEV Protease The TEV protease recognition sequence with the highest catalytic efficiency is ENLYFQ S; however, the amino acid in the P1 position can also be G, A, M, C, or H (1). As your trusted, collaborative partner, Avantor Services helps you increase efficiency, maximize productivity, and accelerate innovation. The weighted average emission wavelengths (nm) for each sample are plotted as a function of Gd-HCl concentration (M). The impact of the F217K mutation was manifest on Km but not on kcat, which suggests that this side chain contributes, either directly or indirectly, to substrate binding. To facilitate the separation of the larger cleavage product from the substrate, we synthesized two new peptides: SP-3246 (TENLYFQSGTRR-NH2) and SP-3247 (PTTENLYFQSGTRR-NH2). The kcat values were calculated by assuming 100% activity for the enzymes. TEV protease self-cleavage was first demonstrated in plant cells, 25 but has only once been utilized to separate two proteins expressed from E. coli cells. There is no need to calculate the molar ratio. VWR provides the cell culture community with access to the most reliable supply of exceptional quality Fetal Bovine Serum: VWR Life Science Seradigm. and Hanna,P.E. Concentration dependence of autoinactivation. Next Generation Sequencing Library Preparation, DNA Assembly, Cloning and Mutagenesis Kits, Supporting Infectious Disease Research & Development. and Koonin,E.V. Cleavage occurs between Q and G. Since the final aa remains on the cleaved protein where it could potentially affect structure or function, substitution of a variety of aa have been tested. Very little is known about the regulation of NIa protease during viral infection, but we note that both the 27 kDa catalytic domain and its 25 kDa autolysis product, which correspond, respectively, to the full-length and truncated HisTEVArg proteases analyzed in this study, have been detected in preparatons of NIa protein isolated from infected tobacco plants and have also been observed to accumulate when the 49 kDa NIa precursor is synthesized in a cell free transcription/translation system (Dougherty and Parks, 1991; Parks et al., 1992,1995). Lys and Asp are the residues that occur most frequently at positions 217 (P2) and 219 (P1), respectively, and so it seemed reasonable to assume that TEV protease would tolerate these amino acid substitutions. WebTEV Protease is a 3C-type protease that cleaves substrates with a consensus sequence of ENLYFQG. In an effort to reduce or abolish autoproteolytic inactivation of TEV protease, we constructed and characterized several mutants with amino acid substitutions adjacent to the internal cleavage site. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. The inactive protease used in this experiment (BAPTEV(D81N)His) has a hexahistidine tag on its C-terminus and a BAP on its N-terminus. Just prior to lysis, phenylmethylsulfonyl fluoride (PMSF) and benzamidine were added to final concentrations of 1 and 2.5 mM, respectively. FOIA WebThe amount of GST-3C protease can be determined by measuring the absorbance at 280 nm of the protein solution against the dialysis buffer. Accordingly, we reasoned that it might be possible to create a protease that is more resistant to autoinactivation by altering the side chains in the P2 and/or P1 sites. A representative plot of initial velocity versus substrate concentration for one of the mutant proteases (S219P) is shown in Figure 6. TEV protease (EC 3.4.22.44, Tobacco Etch Virus nuclear-inclusion-a endopeptidase) is a highly sequence-specific cysteine protease from Tobacco Etch Virus (TEV). After 3 h, the reactions were subjected to electrophoresis in a 12% Trisglycine gel (Novex) and then electrophoretically transferred to a nitrocellulose membrane, using a Novex ExCell II device according to the manufacturer's instructions. Cleavage occurs between Q and G. Since the final aa remains on the cleaved protein where it could potentially affect structure or function, substitution of a variety of aa have been tested. Thus, our results demonstrate that the amino acid sequence in the immediate vicinity of the internal cleavage site contributes to enzymesubstrate recognition in a manner that is consistent with the known specificity of TEV protease, and lend further support to the notion that knowledge about the specificity of a protease can be used to guide the engineering of mutants with a reduced propensity for autoproteolysis (Rose et al., 1993). Unable to load your collection due to an error, Unable to load your delegates due to an error. At pH <5, TEV protease is inactive.1 Under in vitro conditions, native TEV protease has optimal activity in the absence of monovalent It is often used for the removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins. Kinetic parameters were determined for SP-3246. The dimeric form of the viral main protease is responsible for the cleavage of the viral polyprotein in 11 sites, including its own N and C-terminus. Kim Y, Babnigg G, Jedrzejczak R, Eschenfeldt WH, Li H, Maltseva N, Hatzos-Skintges C, Gu M, Makowska-Grzyska M, Wu R, An H, Chhor G, Joachimiak A. et al. 8*(3jc .V'>>VP=Ryi!>BL6.:IPYV.voRD=2^a}K~Z+$ (, Tomasselli,A.G., Paddock,D.J., Curry,K.A., Garlick,R.L., Leone,J.W., Lull,J.M. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. WebSuper TEV extinction coefficient: 31,970 M-1cm-1 at 280 nm van den Berg et al (2006) Improved solubility of TEV protease by directed evolution. and Hartmanis,M. Autoinactivation of TEV protease results in substantial losses during purification of the enzyme, and the truncated product is not easy to separate from the full-length protease. Lanes: 1, HisTEVArg only; 2, BAPTEV(D81N)His only; 3, HisTEVArg + BAPTEV(D81N)His. :T?_isb!0iLV_vm6kfMA&6veo$mvNw=8PA |uow!{/@F14Pa!~@qZ.JB(lB@iVCMp@b`T7[;] wKyf7t}]K^BIzov ;8\R`8.C4A6^]tety 9{,u)q3%k&AmbXC+T6u;$kZh\EDRAW\W5RTbG+rTQTb"g2 9*pfKU9 It may be used in biotechnology to cleave affinity tags from recombinant proteins, either co-translationally or in vitro following purification. (, Southworth,M.W., Amaya,K., Evans,T.C., Xu,M.Q. Incubate at 30C for 1 hour or at 4C overnight. WebI am purifying a GST-tagged protein, and cleaving the tag with TEV protease. (, Ho,S.N., Hunt,H.D., Horton,R.M., Pullen,J.K. Removal of Affinity Tags with TEV Protease. PLoS One. The site is secure. (, Dougherty,W.G., Carrington,J.C., Cary,S.M. Our solutions, developed with you as our focus, are crafted by our team and network of professionals with advanced degrees in science, quality control, engineering, manufacturing and industry experience. Boross,P., Bagossi,P., Copeland,T.D., Oroszlan,S., Louis,J.M. An improved strategy for high-level production of TEV protease in Escherichia coli and its purification and characterization. krMwGke u&J_'">F tx0\,6&;>%:Qwld@osyKiO !*vy{_DqE0[9mL8gfyVF=_jsDVkjs0!5#]$oe{oxp!wvt)r*p!8 SbY,"n%EI-?Q)9b^zkJ$~Re %\_Yz-Up[3M{IyTF"aGx9+GxOg,Z'K7XD=;mV_+.r\BV5eu f =]4l }4A_YM#}P{mZ51ZdYv@~h;&}X/;.eVIz(aeQ4c05V3KAGeYep(6r1Y;7`* ySiI?Q!Z]tzl5 k8X.VN\. The substrate and the cleavage products were separated using an increasing wateracetonitrile gradient (0100%) in the presence of 0.05% TFA. After 15 min on ice, the precipitates were pelleted by centrifugation at 6000 g (10 min at 4C). Typical reaction conditions are as follows: Combine 15 g of substrate and H 2 O (if necessary) to make a 45 l total reaction volume. All of the enzymes were stable in the assay buffer during the studied time interval, as monitored by SDSPAGE (data not shown). To add items to your cart, enter a quantity and click Add to Cart. Epub 2006 Jul 12. The yield and solubility of the F217K and S219D mutants in E.coli was similar to that of wild-type TEV protease, but much less truncated material accumulated during the purification of the mutants, particularly the F217K protease (data not shown). WebPreScission Protease is a fusion protein of glutathione S-transferase (GST) and human rhinovirus (HRV) type 14 3C protease. Disclaimer, National Library of Medicine Because the mutant proteases were intended to be used as reagents for cleaving genetically engineered fusion proteins in vitro, we thought it would be prudent to compare the global stability of the S219 mutants with that of the wild-type protease in an equilibrium denaturation experiment monitored by fluorescence spectrometry (Figure 7). Typical reaction conditions are as follows: Contact your local subsidiary or distributor. My buffer contains 20 mM HEPES, pH 7.5, 300 mM NaCl, 5 mM DTT. and Deutzmann,R. [)3-,:.9y@4(|Z1E3hy~os:;XTxy^o2.M2yN)|G=z'x v0a}!4'Q^j7L"QNco:o_lY 2022 FORTUNE Media IP Limited All rights reserved. }n>=Ce,xV:Xp ,.>"2S]b}t"~.2W)zmwG X&2 {}.eR;.;/ (b^,-~? Cleavage occurs between Q and G. Since the final aa remains on the cleaved protein where it could potentially affect structure or function, substitution of a variety of aa have been tested. Our bioprocessing products are engineered to the highest quality standards and regulatory requirements. In fact, the S219E and S219V mutants were only slightly less resistant to autolysis than the F217K mutant, and the S219P mutant appeared to be virtually impervious to autoinactivation under all conditions. As stated above, only the residues in the P2 and P1 positions of the internal cleavage site in TEV protease match the consensus target sequence; the other positions that are known to influence the efficiency of processing (P7, P4 and P1) are already occupied by unfavorable residues. government site. Oligopeptides were synthesized by standard 9-fluorenylmethyloxycarbonyl chemistry on an Applied Biosystems model 430A automated peptide synthesizer. The reaction products were separated by SDSPAGE on a 12% Trisglycine gradient gel, electrophoretically transferred to a nitrocellulose membrane, and then probed with a streptavidin-horseradish peroxidase conjugate. Intermolecular proteolysis was assayed by incubating biotinylated, catalytically inactive BAPTEV(D81N)His protease alone or together with an equal concentration (67 M) of wild-type HisTEVArg protease in reaction buffer at 30C. 2022 VWR International, LLC. The column was stripped with four volumes of 0.1 M glycine (pH 2.8) between the chromatography cycles. and Dougherty,W.G. Methods Mol Biol. (, Tsao,K.L., DeBarbieri,B., Michel,H. The concentration of HisTEVArg protease stock solutions was determined by amino acid analysis. Therefore, we investigated whether autoinactivation of TEV protease occurs via an intermolecular mechanism by incubating a catalytically inactive form of TEV protease, containing a D81N substitution in the catalytic triad (Dougherty and Parks, 1989), with a catalytically active (wild-type) HisTEVArg protease. Only cleaves a Gly-Gly dipeptide at its own C-terminus ( PubMed: 2656254 ). Present address: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0580, USA, To whom correspondence should be addressed. This vector was constructed by PCR amplification of the ORF encoding the D81N mutant TEV protease contained on pRK580 (R.Kapust, unpublished data) with primers PE-619 (5-GTC GGT CTC GAG CGG AGA AAG CTT GTT TAA GGG GCC GCG T-3) and PE-620 (5-CTC CCT GGA TCC TTA GTG ATG ATG GTG GTG ATG ATT CAT GAG TTG AGT CGC TTC C-3). Explore our solutions for multiple applications - vaccines, monoclonal antibodies, recombinant proteins, cell or gene therapy. After the cells were lysed by sonication, polyethylenimine (Sigma) was added to 0.1% (w/v) and the crude lysate was clarified by centrifugation at 37 000 g for 10 min. in 500 mL erlenmeyer flask Final ampicillin concentration (selects for pRK793): To compare product details, select up to 3 alternatives below and click Compare Selected. VWR supports you in your autosampler vial selection process through on-site consultations with our Chromatography Specialists, and providing samples when needed. An S219V mutation in the protease reduces its rate of autolysis by approximately 100-fold and also gives rise to an enzyme with greater catalytic efficiency than the wild-type protease. Reactions may be scaled-up linearly to accommodate larger sample amounts and reaction volumes. We reasoned that if the amino acid sequence in the immediate vicinity of the internal cleavage site contributes to enzymesubstrate recognition in a manner that is consistent with the known specificity of TEV protease, then replacing Ser219 with Glu, Val or Pro should give rise to proteases with greater resistance to autoinactivation than the S219D mutant. ejr word/document.xml]nH_iY[[v=]=.5& 9@>^{R official website and that any information you provide is encrypted and Perler,F.B. Parks,T.D., Smith,H.A. Freeze cells in tubes from 1 to 5mL using the Thermo Scientific Mr. Frosty Freezing Container at nearly -1C/minute. Your profile has been mapped to an Institution, please sign back for your profile updates to be completed. 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