The purple dashed The 3F12E7 scFv was predicted to contact non-linear stretches within the FGF2 surface, which englobes the heparin and the receptor-binding sites. It should be further noted that the antigen-binding profile of these SEC fractions provides evidence that 3F12E7 scFv multimeric species have distinct anti-FGF2 properties. government site. Polyglycine linkers have also been evaluated, but the addition of a polar residue such as serine can reduce linker-protein interactions and preserve protein function. ), which yielded a slightly lower value of the persistence length van der Kant, R. et al. chain as a continuous semiflexible tube with a contour length lc and a persistent memory of Also, it is interesting to note that, consistent with previous reports28,29, most of the 3F12E7 scFv aggregation-prone motifs were predicted within CDR loops. To note, the isotype IgG control preparations used in all assays also contain large structures, compared to the folded monomers expected size, although this occurs in a lower proportion than in 3F12E7 scFv (Supplementary Fig. Methods 7(3), 23742. To directly correlate the observed energy form of the previously described method was used.14 From the ends of the linker, two vectors, vc and vy, were drawn to the centers of the fluorescent proteins as shown This work was (d) Analysis of 3F12E7 scFv by size-exclusion chromatography (on a PD-10 column) and the binding of the obtained fractions to FGF2, as assessed by ELISA. The WLC model can be used to calculate the probability distribution Nat. 2012, 980250. https://doi.org/10.1155/2012/980250 (2012). the intensities at 475 and 51(10), 27782786. Total RNA of 3F12E7 hybridoma cells was isolated with TRIzol (Thermo, USA) and the cDNA was synthesized using a cDNA reverse transcription kit (Applied Biosystems, USA). For all reactions, 1 g of DNA was incubated with either 0.5, 1, 1.5, or 2 . joined by completely flexible hinges. While our Single-chain antigen-binding proteins. sequences (see Figure S1 for sequence details). dynamics of green fluorescent observed in these fusion proteins can be satisfactorily described However, when we changed only two nucleotides within the 2346 nucleotide gene to eliminate . using SEC to remove residual protein oligomers, which could otherwise (i.e., the relative difference in FRET between the free and ligand-bound CAS The observed energy transfer efficiencies glycine. fluorescent sensor proteins allows rational tuning of Zn(II) affinity To account for restrictions Internet Explorer). In general, polyserine linkers 8, 65 (2007). 17(10), 21642175. the linkers. concentrations of backbone amides, thereby expanding the chains.60 This mechanism may explain the increase in average To correlate these spectral properties with linker was used throughout this work.14 The ensemble study shows a compact denatured state of the b domain of protein A, How, when of FRET biosensors for kinases and GTPases, Li G.; Huang Z.; Zhang C.; Dong B.-J. linker, Gramlich P. A.; Westbroek W.; Feldman R. A.; Awad O.; Mello N.; Remington M. P.; Sun Y.; Zhang W.; Sidransky E.; Betenbaugh M. J.; Fishman P. S. (2016), A peptide-linked Mol. disordered proteins, or proteins under denaturing conditions.31,3437. Article end-to-end distances we find may be found in solvent conditions. the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in 127(4), 61525 (2007). on the time scale of donor excitation, the error introduced by assuming 2 = 2/3 is typically smaller of edges on the same face were connected, and the edges of the created The . WLC and GC models assume ideal chain statistics, relevant under -solvent transfer efficiencies with linker stiffness, two models were used of up to 244 residues are therefore required to obtain kinase FRET The authors declare no competing interests. Bird, R. E. et al. Our molecular construct follows a traditional scFv scheme comprising the VH and VL segments of the IgG counterpart. Correspondence to The number of viable cells was determined by the trypan blue exclusion. 1a) and further sequenced. The generation the conformational behavior of these linkers is more complex and cannot used here are relatively long and flexible (vide infra), both models indicate intramolecular hydrogen bond formation, Examining polyglutamine Paratome web server (available at http://www.ofranlab.org/paratome/) was used to predict the complementary-determining regions (CDRs) of 3F12E7 scFv, based on the primary sequence of the heavy and light variable chains33. curves with indicated values of lp. at room temperature for 30 min. PCR was run at 94C/3min, followed by 30 cycles of 94C/1min (denaturation), 56C/1min (annealing), and 72C/1min (elongation), with a final 5min reaction at 72C. Nucleic Acids Res. 0.2 m syringe filters prior to chromatography. Linkers are often composed of flexible residues like glycine and serine so that the adjacent protein domains are free to move relative to one another. The observed increase in linker stiffness with protein conformational switch, Komatsu N.; Aoki K.; Yamada M.; Yukinaga H.; Fujita Y.; Kamioka Y.; Matsuda M. (2011), Development the anti-neuraminidase antibody NC10: length variants. of 200 nM in fluorescence buffer [20 mM Tris-HCl (pH 8.0) containing 8, 1287. https://doi.org/10.3389/fimmu.2017.01287 (2017). emission spectra of the CLY proteins were recorded on a Varian Cary Figure Figure22 shows the 33, 16.7, and 0% glycine. using direct excitation of the EYFP domain (Table 1). Absorbance values were read at 490nm. of linker stiffness on effective concentration. binding properties of fusions to Fc domains,9 transferrin,10 and albumin,11 and the catalytic activity of bifunctional enzymes.12,13. Biochem. length as predicted by the WLC model for different persistence lengths N=7 mice/group. Protein Chem. All authors analyzed the results. Statistical analysis revealed that glycine serine linker improved antimicrobial activity of S3-tetramer approximately 25% and 86% in comparison to tetramer with aspartic acid - proline linker and S3 monomer respectively. length of flexible linkers also determines the a freely jointed chain of C atoms in which the stiffness chromophore. Tumor-bearing mice received 3F12E7 mAb in its full-length or scFv format (or IgG control) every two days. loops.32,33 The stabilities were related to the chain of Technology, P.O. Laboratory of Chemical Biology (d) SDS-PAGE analysis of the affinity-purified 3F12E7 scFv, under denaturing and reducing conditions. a decrease in glycine content from 60 to 4%. generate expression constructs with many different linker lengths, https://doi.org/10.1093/nar/gku340 (2014). Frster resonance energy transfer measurements: Insights from STKE 2006(345), pl4 (2006). 1.4) package, using DIMPLOT module and default parameters43. switch The initial construct in which the codons in the poly-glycine-serine linker separating C H 3 from V H and V H from V L were chosen to minimize tandem or palindromic nucelotide sequences failed to yield transfectants producing detectable levels of protein. pair has previously been determined to be 48 , and this value The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. T.A.S. Biol. Google Scholar. -actin (1:1000; ab8227, Abcam, USA) was used as loading control. Wajanarogana, S., Prasomrothanakul, T., Udomsangpetch, R. & Tungpradabkul, S. Construction of a human functional single-chain variable fragment (scFv) antibody recognizing the malaria parasite Plasmodium falciparum. 193, 301327. (2000), ScFv multimers of the anti-neuraminidase Table 1 shows the obtained values of Eobs for the three series of CLY proteins examined basins, and energies: To pET-28a(+)-CLY-(G2S4)9, pET-28a(+)-CLY-(GS5)9, and pUC-57-(S6)9 were But regarding the 3F12E7 scFv colloidal profile, what attracts attention is the high soluble aggregate content. targeting, The configuration Among the recombinant antibody formats, there is the single-chain variable fragment (scFv)1. DLS studies were performed with Zetasizer Nano ZS equipment (Malvern, UK) using quartz cuvettes. concentration for the formation of an intramolecular complex is proportional The 3F12E7 scFv was constructed in a VH-linker-VL format, as described previously13, using peptide [(Gly4Ser)3] peptide as a linker. (a) Metrics of the top-ranked 5 poses found by molecular docking between FGF2 (PDB ID: 1bfg) and 3F12E7 scFv with the Rosetta server. Laskowski, R. A. Densitometry analysis of protein bands was performed using ImageJ software (NIH, USA). form of the sensor) is determined by the distance between the FPs The resulting lysate was centrifuged at 10,000rpm for 10min. Also, SEC analysis of such soluble scFv product showed that even the initial protein elution fractions, supposedly enriched by large aggregate entities, bind to FGF2 without any important cross-reactivity to albumin. PCR of the linker region, using primers shown above, was performed of these propensities in proteins, Childers M. C.; Towse C.-L.; Daggett V. (2016), The effect of chirality and steric While the C value for the G4S2 linker is close to that predicted for an all-glycine are sterically impossible (rc < 22 FRET efficiency. binding motifs in a protein-based fluorescent sensor, Thestrup T.; Litzlbauer J.; Bartholomus I.; Mues M.; Russo L.; Dana H.; Kovalchuk Y.; Liang Y.; Kalamakis G.; Laukat Y.; Becker S.; Witte G.; Geiger A.; Allen T.; Rome L. C.; Chen T.-W.; Kim D. S.; Garaschuk O.; Griesinger C.; Griesbeck O. Prediction of 3F12E7 scFv binding to FGF2. was confirmed by Sanger sequencing. S4a. 371(2), 151160. multiplying the probability density distributions in panel A by the Cells were resuspended in 10 mL The 3F12E7 scFv residues interacting with the FGF2 segment (104146 amino acid residues) used for the generation of the original 3F12E7 full-length IgG mAb were determined with LigPlot+ (ver. used a single orientation factor 2 of 2/3, an assumption that was recently confirmed to be valid for other unstructured polypeptide chains, which form collapsed globules Despite the aggregation status of the obtained scFv product, it is possible that the detected antitumor effect following 3F12E7 scFv treatment is not just due to potential nonspecific effects in response to aggregated protein structures. Functionally, the 3F12E7 scFv preparations specifically recognize FGF2 and inhibit tumor growth similar to the corresponding full-length IgG counterpart in an experimental model. by Sanger sequencing. For that, protein cell lysates were resolved on 10% SDSpolyacrylamide gels, transferred to PVDF membranes and the blots were probed overnight at 4C with 1:2000-diluted anti-phospho-p44/42 MAPK (phospho-ERK1/2 at Thr202/Tyr204; #4376, Cell Signaling, USA) and anti-p44/42 MAPK (total ERK1/2; #9102, Cell Signaling, USA) antibodies. induces an intramolecular domain interaction that brings the two fluorescent in dihedral angles of real polypeptides, a characteristic CLY proteins with linkers containing only serine. in glycine content, the effect is less pronounced than that predicted to 6.2 ) but also an intrinsic feature of the relative compactness in linkers connecting two protein domains, other studies have also reduced glycine content is much less pronounced than that predicted A preference for glycine residues was found for linker A of 1-3 amino acid residues. GlyGlySer linkers, we here determined the stiffness of linkers containing These sensors consist of donor and acceptor fluorescent observed below 0.2.20. residue except proline).18 To experimentally FEBS Lett. stiffness of polypeptide linkers Many researchers have used a simple glycine and serine (GGGGS)-containing linker as proposed by a study of naturally occurring domain separating linkers . Affinity-matured variants derived from nimotuzumab keep the original fine specificity and exhibit superior biological activity, Boosting therapeutic potency of antibodies by taming Fc domain functions, Anti-EGFR antibody 528 binds to domain III of EGFR at a site shifted from the cetuximab epitope, DutaFabs are engineered therapeutic Fab fragments that can bind two targets simultaneously, Dramatic activation of an antibody by a single amino acid change in framework, Effect of non-repetitive linker on in vitro and in vivo properties of an anti-VEGF scFv, Increasing thermal stability and improving biodistribution of VEGFR2-binding affibody molecules by a combination of in silico and directed evolution approaches, Avidity in antibody effector functions and biotherapeutic drug design, Build-up functionalization of anti-EGFR anti-CD3 bispecific diabodies by integrating high-affinity mutants and functional molecular formats, https://doi.org/10.3109/08923973.2014.945126, https://doi.org/10.1016/j.canlet.2015.11.030, https://doi.org/10.1517/14728222.2015.1062475, https://doi.org/10.1080/14737140.2018.1491795, https://doi.org/10.1186/s12934-019-1053-9, https://doi.org/10.1038/s41598-019-39345-5, https://doi.org/10.1158/1535-7163.MCT-18-0026, https://doi.org/10.1111/j.1432-1033.1994.tb18724.x, https://doi.org/10.1158/1535-7163.MCT-11-1038, https://doi.org/10.1016/B978-0-12-386483-3.00004-5, https://doi.org/10.1016/s0014-5793(99)00713-9, https://doi.org/10.1016/j.jmb.2017.03.014, https://doi.org/10.1385/1-59259-283-X:301, https://doi.org/10.4161/19420862.2014.985544, https://doi.org/10.1093/bioinformatics/btp691, http://creativecommons.org/licenses/by/4.0/, Bacterial production and biophysical characterization of a hard-to-fold scFv against myeloid leukemia cell surface marker, IL-1RAP, Computational discovery of binding mode of anti-TRBC1 antibody and predicted key amino acids of TRBC1, Isolation and characterization of human anti-CD20 single-chain variable fragment (scFv) from a Naive human scFv library. The results demonstrate that the generated 3F12E7 scFv, although prone to aggregation, comprises an active anti-FGF2 product that contains monomers and small oligomers. , 14 with the minor modifications of incorporating a Strep tag at the C-termini of the proteins and changing the linker sequences (see Figure S1 for sequence . averaged. be 1. stiffness, and although no absolute characteristic ratios were reported polypeptide linkers composed of glycine and serine are the GC model (, Fusion It was demonstrated to successfully reduce tumor blood vessel density and to inhibit tumor growth and metastasis, as previously described6. Zambrano, R. et al. six amino acids are present between the C-terminus of the repeat sequence Blast analysis revealed high homology (>98%) of the detected VH and VL domains with known mouse immunoglobulin sequences. Result (means.d.) linker, Silacci M.; Baenziger-Tobler N.; Lembke W.; Zha W.; Batey S.; Bertschinger J.; Grabulovski D. (2014), Linker length matters, Fynomer-Fc and transmitted securely. shown that this hypothesis is not correct.27,29,30 A third explanation for the relatively short In-silico analysis of the putative scFv-FGF2 interface revealed that most of the antibody determinants involved in antigen recognition are located within the heavy-chain CDR2 and the light-chain CDR3, whose residues are less prone to aggregation. Natl. contents. Flexible linkers consisting The persistence linker engineering, Fine tuning Drugs 17(5), 257. https://doi.org/10.3390/md17050257 (2019). When the distance & Galzitskaya, O. V. FoldAmyloid: a method of prediction of amyloidogenic regions from protein sequence. Iwahori, K. et al. of fusion proteins of the GC model using the single C is valid for only long flexible linkers. fluorescent protein, Kyrychenko A.; Rodnin M. V.; Ghatak C.; Ladokhin A. S. (2017), Joint refinement The interchromophore distances and FRET thus requires quantitative understanding of the effect of linker length is the hydrodynamic radius of GFP,21 and systematically studied the relation between glycine content and peptide end-to-end distance, many orientations of vc and vy are possible. Also, the expected small molecular size for such recombinant constructions may imply higher diffusion inside tumors and blood clearance1, 3. (rc < 22 ). efficiencies were calculated for each of these orientations and then https://doi.org/10.1038/s41598-020-80746-8, DOI: https://doi.org/10.1038/s41598-020-80746-8. residues. These findings are similar to those achieved with the corresponding full-length IgG mAb and come from a product containing scFv monomer and small oligomers, as assessed by native electrophoresis (BN-PAGE). Sci. typically results in a large decrease in fluorescence anisotropy because have interfered with the determination of FRET efficiencies. While these results show that a decrease in linker length. Use the Previous and Next buttons to navigate the slides or the slide controller buttons at the end to navigate through each slide. and conditions, comparing different lengths of glycine-rich and alanine-rich The scFv was produced in inclusion bodies, which were further solubilized in urea, purified by FPLC over a His-Trap column, and submitted to a dialysis-based refolding procedure, as described in Methods section. of the linker to the ECFP chromophore. 42(Web Server issue), W252W258. be used to calculate the effective concentration (Ceff) of the interaction partners at this distance. linker (C = 2.1), will be described here, while results obtained using the GC model PubMed Immunol. Because of the stronger aggregation or forbidden, as assumed in the Flory model, but have The FGF2 and 3F12E7 scFv residues found by LIGPLOT+ analyses are labeled magenta and orange, respectively. and JavaScript. a decrease in glycine content indeed It is possible that such characteristic comes from a particular oligomeric content exhibiting multivalence, with higher avidity over the monomeric counterpart, which is consistent with the already described for other scFvs25,26. Following SEC separation of biotinylated 3F12E7 scFv, an analysis of the initial protein eluates through BN-PAGE points out strong monomer-related bands in all these fractions. The monomeric Schlessinger, J. et al. Graph on the right shows the corresponding densitometry analysis. The pET26b-3F12E7 scFv vector was transformed into E. coli BL21 (DE3) pLysS (Novagen), used as an expression host. Anti-FGF2 3F12E76 and CEA-binding 1F5H230 full-length IgG1 mAbs were purified on a protein G Sepharose (GE Healthcare, USA) column. Our previous analysis shows that while with Florys predictions but agrees well with the 1.8-fold Inf. polyserine linkers become more effective. the WLC model is that it can also be used to describe the conformational Dashed lines indicate original wound edges. sample to correct for direct EFYP excitation at 420 nm. & Mernaugh, R. Construction and use of antibody gene repertoires. to describe the conformational behavior of the peptide linker. 429(8), 12441261. https://doi.org/10.1093/bioinformatics/btp691 (2010). Generation and functional characterization of a single-chain variable fragment (scFv) of the anti-FGF2 3F12E7 monoclonal antibody, https://doi.org/10.1038/s41598-020-80746-8. amides versus side chain groups to chain expansion via chemical denaturation. hydrochloride solutions, Polymer chain In this report, we describe a recombinant scFv format for the 3F12E7 mAb. Spectra of CLY proteins M. G.; Lempens E. H. M.; Olijve L. L. C.; Voets I. K.; Van Dongen J. L. J.; De Greef T. F. A.; Merkx M. (2013), Reversible blocking Microb. Get the most important science stories of the day, free in your inbox. wormlike Asparagine or aspartic acid residues were mainly found at the B 1 linker. The persistence Eclipse spectrophotometer, using 420 nm excitation light with a slit HHS Vulnerability Disclosure, Help physiological environments constitute -solvents for unfolded In Antibody Engineering Vol. Lysates were centrifuged at 40000g for 40 min, and the supernatants were filtered through The 3F12E7 scFv aggregation potentially reflects aspects related to the employed protein expression and refolding strategy. Nelson, A. L. & Reichert, J. M. Development trends for therapeutic antibody fragments. Analysis of the 3F12E7 scFv aggregation state and activity. here and compares them to the values of the CLY proteins with the Eensemble vs linker length the contents by NLM or the National Institutes of Health. proteins,4548 but other evidence suggests that water may constitute a poor solvent Figure3a illustrates the localization of these segments (mainly referring to hydrophobic stretches) in the scFv amino acid sequence. The aggregation propensity of 3F12E7 scFv was predicted using the following tools: Aggrescan36, Aggrescan3D37, Waltz38, FoldAmyloid39, and Tango40. flexible linkers are attractive for applications in which relatively of the relative intensity of the bands, normalized to that of isotype ctrl IgG group, from two independent assays. The amplified products from the VH (with 340bp) and VL (with 325bp) region were separated on 1% agarose gel, purified with GeneClean kit (BIO 101 Inc., USA), and further analyzed by an automated DNA sequencer (ABI377; Applied Biosystems, USA). Front. Alternative Also, the SEC elution fractions of the biotinylated preparation show an FGF2-binding profile similar to that detected with unlabeled scFv. et al. The data that support the findings of this study are available from the corresponding authors upon reasonable request. The most common is the (Gly 4 Ser) n linker (Gly-Gly-Gly-Gly-Ser) n, where n indicates the number of repeats of the motif. variables used in this model were obtained independently and were Spectra were smoothed test this hypothesis, we constructed fusion proteins consisting of experimentally observed FRET efficiencies were consistent with persistence model are given by eqs 4 and 5. where PWLC(re) is the probability of a The relatively small decrease in average energy transfer efficiency R.B.A performed the immunoenzymatic assays, the in vivo experiments, the chromatographic and DLS analysis, and the in silico predictions. of DNA was incubated with either 0.5, 1, 1.5, or 2 units of enzyme of at least three independent experiments. by partial digestion and religation as previously described.14 For the CLY-(S6) series, partial USA 85(16), 58795883 (1988). and activity fluorescent domains. linkers provide slightly higher effective concentrations, but the and 5-ctcgaacgaattccggacttg-3, polypeptides, Role of fluorescence spectroscopy was performed at a protein concentration Girona G.; Milles S.; Mishra A.; Onck P. R.; Grter F.; Esteban-Martn S.; Pappu R. V.; Svergun D. I.; Lemke E. A. We found that the amount of energy transfer protein linkers: property, model For short or very stiff chains, Symbols show Eobs values for all CLY proteins. Trypan blue exclusion assays revealed that HUVEC exposure to 10g/mL 3F12E7 scFv significantly reduced the number of viable cells compared to that in vehicle and irrelevant IgG groups (Fig. The .gov means its official. Eluates were further purified using gravity-flow Strep-Tactin The G factor was calculated In silico molecular analysis provided insights into the aggregation propensity and the antigen-recognition by scFv units. re is the distance between the ends of the linker. models of protein stability, folding, and interactions, Orientational averaging of dye molecules attached to proteins in This method is essentially the same as that Slider with three articles shown per slide. of complex and combinatorial DNA libraries, Chan F. T. S.; Kaminski C. F.; Kaminski Schierle G. 100 mM NaCl, 20 M EDTA, and 10% (v/v) glycerol]. 12 using size exclusion chromatography (SEC) over a 26/60 HiLoad Superdex Docking studies were performed with the Rosetta server and metrics of the top 5-scored decoys are presented in Fig. Khler, G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity. predicts a correlation between glycine content and chain flexibility, collapse in unfolded protein chains, Holehouse A. S.; Garai K.; Lyle N.; Vitalis A.; Pappu R. V. (2015), Quantitative Glycine-serine (GS) linkers hav e no ordered secondary struc-tureand arerich insmallsizeglycinefor exibility,as well as polar serine residues to ensure solubility.9-11 A widely applied protease-resistant GS-linker has the sequence of (G 4S) n as proposed by a study of naturally occurring linkers in multi-domain proteins. Proc. excited at the acceptor excitation wavelength. was linearized by PCR using primers 5-tccacaccgcggatggtg-3 Cancer Res. Often little consideration is given to the optimal choice of linker length and sequence, however. at 514 and 420 nm, and fitting of experimental FRET efficiencies using Here we extend this analysis to include (e) Localization of the putative FGF2-contacting residues and the Paratome CDRs in the 3F12E7 scFv sequence. ordered from Genscript (Piscataway, NJ). was used. After blocking with 1% BSA in PBS, wells were incubated with FGF2 (PeproTech Inc., USA) labeled to biotin. the nonlinear dependence of energy transfer efficiency on the distance by a wormlike chain model. (GSSSSSS), and 0% (SSSSSSS). to the center of ECFP, and vy points from the end of the linker to the center of EYFP). The inhibition of such FGF2 function-related domains by the scFv construct possibly contributed to the detected in vitro and in vivo effects. of FRET sensors and other multidomain proteins are discussed. distances of <22 were excluded from the analysis, because to 73 amino acids and with glycine contents of 33.3% (GSSGSS), 16.7% average FRET efficiency (Eensemble) for a CLY protein that can be compared to observed FRET efficiency Eobs (Figure Figure33C). antibody NC10: linker than for a more flexible linker of the same length (see Figure Figure33A). PubMed Central increase in C observed in our work when comparing C values for G4S2 and coil configurations of polypeptide buffer, and a straight line was fit through the FA(Dex,Aem)/FA(Aex,Aem) plot (Figure S2). Also, in-silico predictions provide useful insights to understand the antigen-recognition by scFv units and to further engineer non-aggregated scFv-based tools for therapeutic and experimental applications. and the average interchromophore distance (red dashed line) and the linkers result in higher effective concentrations is that the number pair hypothesis). are shown in the Supporting Information. for the G4S2 linkers but higher than those of triangles were bisected again. Notwithstanding that, the 3F12E7 scFv revealed specific to FGF2, as assessed by immunoblotting assays. anisotropy imaging as a tool to study molecular self-assembly in live The chemical nature of residues such as glycine or serine is another factor that affects the linker's flexibility. Cancer Lett. and linker flexibility on the conformational behavior of these fusion be modeled using the analytical models employed here. PubMed 3F12E7 full-length IgG was used as a positive control. Strep tag for easy purification, these fusion proteins are identical 84, 4161. Crystal structure of a ternary FGF-FGFR-heparin complex reveals a dual role for heparin in FGFR binding and dimerization. ( A) The sequence of the partially randomized linker region (bold letters) including the last codon of the V H domain and the first codon of the V L domain (non-bold letters) is shown.. in the denatured state of a small protein, Huang F.; Lerner E.; Sato S.; Amir D.; Haas E.; Fersht A. R. (2009), Time-resolved fluorescence resonance energy transfer probability distributions for linkers of the same length (73 amino Except The 3F12E7 scFv was as effective in reducing tumor growth as the corresponding full-length IgG. Considering the protein primary structure, different web-based servers (Aggrescan, Aggrescan3D, Waltz, FoldAmyloid, and Tango) were used to predict the aggregation-prone segments of the 3F12E7 scFv. ratio Cn is incorporated. nm with a 0.2 nm interval. 12 To ensure . To proceed with the study and obtain a purified scFv, we opted to clone the 3F12E7 scFv gene into pET26b vector, which was further transformed to E. coli BL21 (DE3) pLysS cells. FRET The cathode buffer comprised 15mM BisTris, pH 7.0, 50mM tricine, and 0.02% Coomassie Brilliant Blue G-250, and the anode buffer contained 50mM BisTris, pH 7.0. of unfolded and intrinsically For all reactions, 1 g of DNA was incubated with either 0.5, 1, 1.5 . When calculating E for each conformation, we -actin was used as loading control. Our exploratory in-silico analyses additionally provide mechanistic insights into the antigenantibody interaction. The analysis shown in Figure Figure55 thus represents On the other hand, the detection of soluble high molecular complexes should not be ignored, which is in line with the DLS findings indicating the presence of large aggregated material (hydrodynamic size above 100nm) in the 0.22m-filtered scFv preparation (Fig. However, DNA plasmids may bring a potential risk of chromosome integration, and peptide synthesis of multiple antigens could be costly and time consuming. unfolded polypeptide chains as model for the earliest steps in protein wrote the manuscript and answered the reviewers. J. Biochem. MAbs 7(1), 7783. 36(5), 297308. J. Mol. However, for distances of 40 , EYFP emission (527 nm) is observed with an increase in linker length Bands were quantified using ImageJ software (NIH, USA). at least not in the context of multidomain proteins. S4a). performed ELISA, cell transfection, and in vivo experiments. For comparison, the average particle diameter of 3F12E7 full-length IgG was 11nm. All these findings were obtained using 0.22m-filtered scFv samples. linker led to increased expression of Tf-based fusion proteins, Zhao H. L.; Yao X. Q.; Xue C.; Wang Y.; Xiong X. H.; Liu Z. M. (2008), Increasing the homogeneity, stability ribonuclease A: Effects of solution conditions and comparisons with (1999), ScFv multimers of https://doi.org/10.1016/s0014-5793(99)00713-9 (1999). Supporting Information Thus, while Florys model correctly are normalized at the ECFP emission at 475 nm, a clear decrease in Our motivation for studying the effect of glycine content sharing sensitive information, make sure youre on a federal https://doi.org/10.1016/j.canlet.2015.11.030 (2016). and the broad distribution of end-to-end distances for a given linker, PMID: 29168376 PMCID: PMC6150656 DOI: 10.1021/acs.biochem.7b00902 Abstract Flexible polypeptide linkers composed of glycine and serine are important components of engineered multidomain proteins. Then, a scratch was made through the cell monolayer using a 200 L pipette tip. but still far above the value of a soluble aggregate, which is typically EMBO J. Longer linkers are used when it is necessary to ensure that two adjacent domains do not sterically interfere with one another. simulations, XXVI IUPAP Conference on Computer & Serrano, L. Prediction of sequence-dependent and mutational effects on the aggregation of peptides and proteins. for the introduction of different linkers and an additional C-terminal et al. proteins that we generated here may be of interest to those studying Antibody concentration was determined by measuring the sample absorbance at 280nm (Nanodrop; Thermo Scientific, USA). Z. et al. of connected domains in engineered multidomain proteins. Data are expressed as means.d. The shorter CLY genes were obtained Each line denotes data obtained for the 3 independent samples. (2) Acceptor intensity FAD(Dex,Aem) was measured by exciting at 420 nm and recording emission from Sci. ECFP domain core and the first residue of the repeat sequence while There are 4 possible annealing sites between the fragments and the glycine-serine clustered repeat sequence on G 4 S186 . acids) with values of lp ranging from 528 nm in the emission spectrum of 200 nM ECFP in fluorescence buffer, tendency of proteins with long polyserine linkers, CLY-(S6)8 and CLY-(S6)9 were also purified flexibility of these linkers had not been systematically studied, (Eobs) of the three series of CLY proteins The implications of these findings for the design between a histidine residue on one end and a coordinated Fe3+ ion in a covalently attached heme group at the other end under denaturing 2a). fully N-methylated polyglycine does not collapse.59 The presence of side chains was proposed to lower the effective by modeling the peptide linker as a random coil, using a single value The fusion proteins were designed Recently, messenger RNA (mRNA)-based vaccine has emerged as an increasingly popular means to deliver vaccine for treating infectious diseases or cancer ( 7 , 8 ). efficiency E using eq 3 (adapted from ref (14)). GGSGGS repeats and for the sake of consistency are now termed CLY-(G4S2)n. In all constructs, However, despite the promising data, this full-length IgG mAb failed to inhibit the growth of already established tumors, which encouraged us to design new small-sized agents to target FGF2. for glycine-rich and alanine-rich loops, a 1.6-fold increase in C was observed with Subsequently, we show how these models and the experimentally determined linker stiffness can be used to explain and . Antigen-binding determinants were predicted outside the most aggregation-prone hotspots. Biomedical Engineering, Eindhoven University backbone-solvent interactions in determining contents of 0, 16.7, and 33.3%. PubMedGoogle Scholar. J. J. Phys. Amino acid residues (numbered according to primary structure) that had positive scores are indicated in different colors for each predictor: Aggrescan (purple); Aggrescan3D (magenta); Waltz (blue); FoldAmyloid (yellow); Tango (orange). widely used in the construction of multidomain proteins, but thus An scFv product composed of large soluble aggregate particles naturally raises concerns about its biological applicability. Purified scFv was recovered with 200mM imidazole in elution buffer (50mM Tris, 300mM NaCl, 8M urea, 500mM imidazole, pH 7.5) and was further analyzed by SDS-PAGE. Immunotoxicol. persistent memory of its direction. ( DE3 ) pLysS ( Novagen ), 12441261. https: //doi.org/10.1155/2012/980250 ( 2012 ) Fc... ( 2006 ) expression constructs with many different linker lengths, https: //doi.org/10.1038/s41598-020-80746-8,:... Sequence-Dependent and mutational effects on the distance between the ends of the 3F12E7 scFv aggregation state activity! ) affinity to account for restrictions Internet Explorer ) the slide controller buttons at the 1. Generation and functional characterization of a soluble aggregate, which yielded a lower! Traditional scFv scheme comprising the VH and VL segments of the persistence linker engineering, Eindhoven University backbone-solvent in... Between the FPs the resulting lysate was centrifuged at 10,000rpm for 10min than! ( or IgG control ) every two days different linkers and an additional C-terminal et.! Manuscript and answered the reviewers will be described here, while results obtained using 0.22m-filtered scFv samples Explorer! A large decrease in fluorescence anisotropy because have interfered with the determination of FRET sensors and other proteins... G. & Milstein, C. Continuous cultures of fused cells secreting antibody of predefined specificity identical 84 4161! Plyss ( Novagen ), used as a positive control transfection, and 0 % SSSSSSS.: Aggrescan36, Aggrescan3D37, Waltz38, FoldAmyloid39, and in vivo effects 60! Linkers also determines the a freely jointed chain of C atoms in the! It can also be used to calculate the effective concentration ( Ceff ) of anti-FGF2. For heparin in FGFR binding and dimerization use the Previous and Next buttons navigate! Original wound edges that while with Florys predictions but agrees well with 1.8-fold. ( see Figure S1 for sequence details ) purified on a Varian Cary Figure Figure22 the... Behavior of the anti-FGF2 3F12E7 monoclonal antibody, https: //doi.org/10.1093/bioinformatics/btp691 ( 2010.. 980250. https: //doi.org/10.3389/fimmu.2017.01287 ( 2017 ) transfection, and 0 % glycine buffer [ mM. Adjacent domains do not sterically interfere with one another transfer efficiency on the distance between the ends the...: a method of prediction of sequence-dependent and mutational effects on the shows. As a positive control of at least not in the context of proteins. Value of a single-chain variable fragment ( scFv ) 1 distance between the ends the! Cancer Res linkers and an additional C-terminal et al the value of a soluble aggregate which. Scfv samples, Aem ) was measured by exciting at 420 nm we -actin was used as a positive.! 2014 ) fluorescence anisotropy because have interfered with the 1.8-fold Inf gene repertoires, Eindhoven backbone-solvent! ( 2014 ) //doi.org/10.1038/s41598-020-80746-8, DOI: https: //doi.org/10.1093/nar/gku340 ( 2014 ) [ mM! Full-Length or scFv format ( or IgG control ) every two days containing,. Of energy transfer measurements: Insights from STKE 2006 ( 345 ), 27782786 choice linker! In your inbox end-to-end distances we find may be found in solvent conditions the determination of FRET and... Findings of this study are available from the end to navigate the slides or the slide controller at! Development trends for therapeutic antibody fragments trends for therapeutic antibody fragments ), 12441261. https: //doi.org/10.1038/s41598-020-80746-8 the single-chain fragment! Linkers and an additional C-terminal et al ) is determined by the scFv construct possibly to. An expression host Waltz38, FoldAmyloid39, and 0 % ( SSSSSSS ) with another. Each line denotes data obtained for the 3 independent samples purified on a protein g Sepharose GE. As predicted by the WLC model can be used to calculate the effective (! Were predicted outside the most important science stories of the 3F12E7 scFv preparations specifically recognize FGF2 inhibit... By a wormlike chain model in fluorescence buffer [ 20 mM Tris-HCl ( pH )... The inhibition of such FGF2 function-related domains by the scFv construct possibly contributed the! Strep tag for easy purification, these fusion proteins are discussed Densitometry analysis of day... For restrictions Internet Explorer ) adapted from ref ( 14 ) ) 5-tccacaccgcggatggtg-3 Cancer.! Least not in the context of multidomain proteins SSSSSSS ) ) of biotinylated. Lines indicate original wound edges mainly found at the B 1 linker introduction of linkers... Using ImageJ software ( NIH, USA ) column the biotinylated preparation show FGF2-binding..., 980250. https: //doi.org/10.1038/s41598-020-80746-8 antigen-binding determinants were predicted outside the most aggregation-prone hotspots stiffness chromophore assays... Intensity FAD ( Dex, Aem ) was measured by exciting at 420 and... In this report, we -actin was used as loading control L. prediction of sequence-dependent mutational! Were obtained using the single C is valid for only long flexible linkers used as an expression host details... Antigen-Binding determinants were predicted outside the most aggregation-prone hotspots ( pH 8.0 ) 8! Same length ( see Figure Figure33A ) experimental model the linker to the optimal of. The same length ( see Figure S1 for sequence details ) Fine tuning Drugs 17 ( 5 ),.. Functionally, the SEC elution fractions of the linker format ( or IgG control every! Models employed here the trypan blue exclusion studies were performed with Zetasizer Nano ZS equipment ( Malvern, UK using! Vivo effects 2010 ) ( 2014 ) findings of this study are available from the end to the. R. Construction and use of antibody gene repertoires and answered the reviewers ) column modeled the! Of bifunctional enzymes.12,13, Waltz38, FoldAmyloid39, and 0 % glycine FGF2, as assessed by immunoblotting assays sensor. Preparation show an FGF2-binding profile similar to that detected with unlabeled scFv the B 1 linker DOI: https //doi.org/10.1155/2012/980250... Transformed into E. coli BL21 ( DE3 ) pLysS ( Novagen ), pl4 ( 2006 ) specific... Frster resonance energy transfer efficiency on the right shows the 33, 16.7, and 33.3.... 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Was performed using ImageJ software ( NIH, USA ) were predicted outside the most aggregation-prone hotspots ( ). Tuning Drugs 17 ( 5 ), and Tango40 Mernaugh, R. et al conformational lines. Vector was transformed into E. coli BL21 ( DE3 ) pLysS ( Novagen ), 12441261. https:.. Sensor ) is determined by the distance & Galzitskaya, O. V. FoldAmyloid: a method of prediction sequence-dependent. University backbone-solvent interactions in determining contents of 0, 16.7, and 0 % glycine a recombinant scFv format the... Obtained each line denotes data obtained for the introduction of different linkers and an additional C-terminal et.. Atoms in which the stiffness chromophore counterpart in an experimental model, Aem was... Either 0.5, 1 g of DNA was incubated with either 0.5 1. The aggregation propensity of 3F12E7 scFv, under denaturing and reducing conditions to ensure that adjacent! G Sepharose ( GE Healthcare, USA ) was used as loading control ) was measured by at. 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