Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-2

to the human immunoglobulins sequences (imgt). Sequences highlighted in red vary from the amino-acid sequence of scFv C1. Underlined sequences correspond to CDR. Asterisks indicate the missing amino-acid that would generate the usual CDR.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-0

sequence of bacterial pectate lyase that mediates secretion into the periplasmic space; : variable fragment of the heavy chain; : light chain; : 6 histidine-tag; : myc-tag; amber stop codon; : portions of the N- and C-term of phage capside protein pIII; and : primers used for sequencing the Vand Vdomain. Schematic construction of vectors encoding soluble scFv C1 and scFv C1-N1N2. The amber stop codon between the scFv and gene III in pHEN 2 was removed by mutagenesis (middle construct). The C-terminal portion of pIII was removed in the final pHEN C1-N1N2 vector (bottom construct), first by PCR amplification of pHEN C1-pIII, introducing an EcoRI site after N2, and subsequently by cloning the NcoI and EcoRI digested PCR product into the linearized pHEN C1-pIII plasmid at the NotI and EcoRI sites. : 6 histidine-tag; M: myc-tag.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-6

recombinant RhoA and RhoB loaded with either GTP or GDP were incubated with C1-N1N2 fixed on Ni-beads. An irrevelant scFv was used as control. Complexes on beads were resolved by SDS-PAGE and immunobloted with anti-RhoA and anti-RhoB. Western Blot is representative of 2 independent experiments. Immunofluorescence shows that scFv C1-N1N2 specifically binds to activated HeLa cells. Suspension containing scFv C1-N1N2 was incubated with GDP-loaded RhoA(and ) or GTPγS-preloaded RhoA beads (). Twenty-four hours after seeding, HeLa cells were serum-starved for 48 h and activated with 10% SVF and EGF (100 ng/ml) for 1 hour. Cells were fixed, permabilized and incubated with supernatants from scFv C1-N1N2-Rho incubation and anti-c-myc FITC conjugate secondary antibody. ) Non-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads, ) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GDP-loaded RhoA beads ) EGF-activated HeLa cells incubated with the antibody scFv C1-N1N2 preincubated with GTPγS-loaded RhoA beads. () Non-activated HeLa cells incubated with the commercial Rhoa antibody, () EGF-activated HeLa cells incubated with the commercial RhoA antibody, () EGF-activated HeLa cells incubated with irrelevant scFv (anti-tyroglobulin). Pictures are representative of 2 independent experiments.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-7

sequence of bacterial pectate lyase that mediates secretion into the periplasmic space; : variable fragment of the heavy chain; : light chain; : 6 histidine-tag; : myc-tag; amber stop codon; : portions of the N- and C-term of phage capside protein pIII; and : primers used for sequencing the Vand Vdomain. Schematic construction of vectors encoding soluble scFv C1 and scFv C1-N1N2. The amber stop codon between the scFv and gene III in pHEN 2 was removed by mutagenesis (middle construct). The C-terminal portion of pIII was removed in the final pHEN C1-N1N2 vector (bottom construct), first by PCR amplification of pHEN C1-pIII, introducing an EcoRI site after N2, and subsequently by cloning the NcoI and EcoRI digested PCR product into the linearized pHEN C1-pIII plasmid at the NotI and EcoRI sites. : 6 histidine-tag; M: myc-tag.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-4

ScFvs were resolved on 12,5% SDS-PAGE, immunoblotted with c-myc antibody, and visualized by enhanced chemiluminescence as described in Methods. (2) scFvs from crude extract were analysed for binding to GST-RhoA and GST-RhoAQ63L protein immobilized on an ELISA plate. Bound scFvs were detected with horseradish peroxydase-labeled anti-c-myc using TMB as substrate. Results are expressed as absorbance at 480 nm. Graphs are representative of 6 experiments, each performed in duplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-3

100 μM of GDP and GTPγS at 37°C. 10clones of C1 phages were incubated with loaded (GTP or GDP) GST-RhoA-bound beads. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. The graph is representative of 2 independents experiments. GST-RhoA, B and C () and GST-RhoB, Rac1 and Cdc42 () were loaded with 200 μM of GDP or GTPγS for 30 min and purified on glutathione ELISA plates. 10clones of C1 phages were incubated in each well. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. Amount of GST protein was quantified with goat anti-GST antibody followed by horseradish peroxydase-labeled anti-goat (not shown). The graph is representative of 3 experiments, each binding assay performed in duplicate. : Specific radioactivity binding of [S] GTPγS on RhoB, Rac1 and Cdc42. GST-RhoB, Rac1 and Cdc42 were loaded with 20 nM [S] GTPγS in the presence (non specific binding) or not (total binding) of 200 μM unlabeled GTP for 30 minutes at 37°C and purified on gluthatione ELISA plates. Radioactivity was measured in each well. The difference between the total binding and the non specific binding represent the specific binding. The graph is representative of 2 experiments, each performed in triplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection-1

RhoB (white columns) and GST-RhoBQ63L (black columns) protein immobilized on an ELISA plate. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Helper phage was used as a control. Results are expressed as absorbance at 480 nm. : ratio of absorbance of binding to RhoBQ63L to absorbance of binding to RhoB. : Selectivity of C1 phages on WT and activated Q63L form of Rho. 10clones of C1 (black columns) and control (white columns) phage were analyzed for binding to GST-RhoA, RhoB and RhoC, both wild type (WT) and Q63L forms, immobilized on a glutathione ELISA plate. Bound phages were detected with horseradish peroxydase-labeled anti-M13 using TMB as substrate. Results are expressed as absorbance at 480 nm. Concentrations of GST-Rho proteins in each well were monitored by anti-GST (not shown). The graph is representative for 3 experiments, and each binding assay was performed in duplicate.<p><b>Copyright information:</b></p><p>Taken from "Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection"</p><p>http://www.biomedcentral.com/1472-6750/8/34</p><p>BMC Biotechnology 2008;8():34-34.</p><p>Published online 31 Mar 2008</p><p>PMCID:PMC2323369.</p><p></p

ScFvE3 preferentially binds to RhoB active conformation.

<p>A, Binding affinities of two selected scFvs (A5 and E3) for 6xHis-RhoAL63 (AL63), 6xHis-RhoBL63 (BL63) were measured by competitive ELISA on dilution of 6his-RhoL63 displayed in molar logarithmic scale (Log M), as described in experimental procedures. K_d values were determined by nonlinear regression and listed in the insert table (mean ± SD, n = 3 each). B, The specificity of the purified scFvE3 for the active form of the recombinant wild type GST-RhoB loaded with either GDP or GTPγS was assessed by ELISA. Results are expressed as absorbance at 450 nm (mean ± SD, Mann-Whitney test, n = 4).</p

scFvE3 is a selective sensor of RhoB activation in HeLa cells.

<p>A, CBD-pulldown experiments on nucleotides loaded HeLa cell extracts showing the specificities of the selected scFvs. HeLa cell extracts were loaded with either GDP (1 mM) or GTPγS (100 µM) and incubated with scFvs F7, D10 and E3 fixed on chitin beads. CBD-pulldowns were analyzed by Western blotting using anti-RhoA, anti-RhoB and anti-RhoC antibodies. Total extract used for CBD-pulldown is indicated as <i>input</i> and examined by western blotting with the same antibodies. Western Blot is representative of 4 independent experiments. B, RhoB and RhoA activation were assessed by GST-pulldown (RBD) and CBD-pulldown (E3) experiments with cell lysate from HeLa cells transiently transfected with plasmids expressing Myc-tagged XPLN or GFP under the control of CMV promotor. XPLN was detected by using an anti-c-myc antibody. C, HeLa cells were serum-starved for 24 h and treated with EGF (2.5 ng/mL) for 10 min before lysis then RhoB and RhoA activation were assessed by GST-pulldown (RBD) and CBD-pulldown (E3) experiments. Beads-bound proteins were analyzed by Western blotting using anti-RhoA and anti-RhoB antibodies. Total cell extracts are indicated as <i>input</i> and examined by western blotting with the same antibodies. Western Blots are representatives of 2 independent experiments.</p

Affinity maturation revealed the possibility to obtain binders distinguishing RhoA and RhoC from RhoB active conformations.

<p>A, Improvement in apparent affinity throughout the rounds of selection was evaluated by a polyclonal phage ELISA on dilution of GST-RhoAL63 displayed in molar logarithmic scale (Log M). B, Three purified scFvs (F7, H9 and D10) were analyzed for their binding specificity towards L63 active mutants of recombinant GST-RhoA, RhoB and RhoC by ELISA. Purified scFvC1 was used as a control. C, Affinities of two scFvs (F7 and D10) for 6xHis-RhoAL63 (AL63), 6xHis-RhoBL63 (BL63) were measured by competitive ELISA as described in experimental procedures. K_d values were determined by nonlinear regression and listed in the insert table (mean ± SD, n = 3 each). D, The specificity of purified scFvF7 and scFvD10 for the active form of the recombinant wild type GST-RhoA and GST-RhoB loaded with either GDP or GTPγS were assessed by ELISA. Results are expressed as normalized absorbance of the scFvs to the total amount of coated GST-Rho quantified by the use of commercial antibodies (mean ± SD, Mann-Whitney test, n = 4 each).</p