Supplementary Information. Pharmacologically Controlled Protein Switch for ON-OFF Regulation of Growth Factor Activity

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1 Supplementary Information Pharmacologically Controlled Protein Switch for ON-OFF Regulation of Growth Factor Activity Maria Karlsson 1,3, Balder Rebmann 1,2, Philipp S. Lienemann 4,5, Natallia Sprossmann 1,2, Martin Ehrbar 4, Gerald Radziwill 1,2 and Wilfried Weber 1,2,3 * 1 Faculty of Biology, University of Freiburg, Schänzlestrasse 1, Freiburg, Germany 2 BIOSS Centre for Biological Signalling Studies, University of Freiburg, Schänzlestrasse 18, Freiburg, Germany 3 Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, 4058 Basel, Switzerland 4 Department of Obstetrics, University Hospital Zurich, Schmelzbergstr. 12, 8091 Zurich, Switzerland 5 Institute of Bioengineering, Ecole Polytechnique Fédérale de Lausanne, Station 15, Bld AI 1109, 1015 Lausanne, Switzerland * To whom correspondence should be addressed: Phone, ; Fax, ; wilfried.weber@biologie.uni-freiburg.de

2 Supplementary Methods Protein production The expression vectors plmk550 and plmk619 were transformed into Escherichia coli BL21* (DE3) plyss (Invitrogen, Carlsbad, CA, cat. no. C ). The protein production was induced at an OD 600 of 0.6 by the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG, Carl Roth, Karlsruhe, Germany, cat. no. CN08.3) to a final concentration of 0.5 mm. After 2 hours at 37 C, the cells were harvested by centrifugation at 6000 x g for 5 min at 4 C, resuspended in lysis buffer (50 mm NaH 2 PO 4, 300 mm NaCl, 10 mm imidazole, ph 8.0) and disrupted using a French press (APV, Albertslund, Denmark, model APV-2000). Cell debris were removed by centrifugation at x g for 20 min at 4 C and the cell lysate was loaded onto a Ni 2+- NTA Superflow column (Qiagen, Hilden, Germany, cat. no ). The column was washed twice with 20 ml wash buffer (50 mm NaH 2 PO 4, 300 mm NaCl, 20 mm imidazole, ph 8.0), after which the protein was eluted in 10 ml of elution buffer (50 mm NaH 2 PO 4, 300 mm NaCl, 250 mm imidazole, ph 8.0). Additionally, for the VEGF SWITCH, the buffer was changed to 0.1 M Tris-HCl (ph 8), the protein was concentrated to 10 mg ml -1 by ultracentrifugation (10 kda MWCO, Corning, Lowell, MA, cat. no ), and the free cysteines were alkylated. For alkylation 0.16 mmol iodacetamide (IAA, Acros, Geel, Belgium, cat. no ) per mg of protein were added, after which the sample was flushed with argon for 30 s and incubated for 1 hour in the dark at room temperature. Finally, for both proteins, the buffer was changed to 50 mm Tris-HCl (ph 7.6) by ultracentrifugation. The VEGF GyrB protein was shock frozen in liquid nitrogen immediately after the medium exchange and stored at -80 C for later use, whereas VEGF SWITCH was subjected to size exclusion chromatography prior to freezing. VEGF 1, 2 as well as Gyrase B 3 were produced as described previously and stored at -80 C. The proteins were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 12%, w/v) under reducing conditions

3 and detected either by Commassie blue or by Western blotting using an anti-vegf antibody (1:1000, Rockland, Immunochemicals, Gilbertsville, PA, cat. no C23) followed by an HRP-conjugated anti-rabbit antibody (1:10000, Santa Cruz Biotechnology Inc., Santa Cruz, CA, cat. no. sc-2004). The VEGF proteins were quantified via a VEGF enzyme-linked immunosorbent assay (ELISA) (Peprotech, Hamburg, Germany, cat. no. 900-K10) according to the manufacturer s protocol and GyrB was quantified by Bradford assay (Biorad, München, Germany, cat. no ) with BSA as standard. Size exclusion chromatography The monomeric fraction of the VEGF SWITCH was isolated by size exclusion chromatography using an ÄKTAprime plus (GE Healthcare, Freiburg, Germany) fast liquid chromatography system with a HiLoad 16/60 Superdex 200 column (GE Healthcare, Freiburg, Germany, cat. no ) equilibrated with 50 mm Tris-HCl (ph 7.6). Size exclusion was carried out at 4 C with a flow rate of 1 ml min -1 and a fraction volume of 2.5 ml. The monomeric fraction of VEGF SWITCH was eluted at ml, which corresponds to a molecular weight of kda. The molecular weight profile was determined using a gel filtration standard (Bio- Rad, Hercules, CA, cat. no ). The isolated protein fraction was resolved by SDS- PAGE (12%, w/v) under non-reducing conditions and quantified via VEGF ELISA. The protein was shock frozen in liquid nitrogen and stored at -80 C.

4 SI Figure 1. Construction and production of the VEGF SWITCH. (a) The amino acid sequence of the VEGF SWITCH. The two cysteine residues in the human vascular endothelial growth factor isoform 121 (VEGF) on positions 51 and 60 responsiblee for the interchain disulfide bridges were replaced with alanine residues (red, bold, italics, underlined). Subsequently, the engineered version of VEGF (red, bold, italics) was fused via a glycineserine rich (black, underlined) linker to the N-terminal domain of gyrase B (green, bold) from Escherichia coli followed by a hexahistidine tag (blue, italics). (b) Western blot analysis of the VEGF SWITCH. The VEGF SWITCH was produced in E. coli BL21* (DE3) plyss, purified by Ni 2+ NTA chromatography and visualized by Western blotting using an anti-vegf 121 antibody 4. (c) VEGF SWITCH isolation. The free cysteine residues of the VEGF SWITCH were alkylated by iodoacetamide and the monomeric fraction of the protein was isolated using size exclusion chromatography. The protein was analyzed by SDS-PAGE on a 12% polyacrylamide gel under non-reducing conditions and stained with Coomassie Brilliant blue. Supplementary Figures

5 Supplementary References 1. Ehrbar M, Djonov VG, Schnell C, Tschanz SA, Martiny-Baron G, Schenk U, et al. Celldemanded liberation of VEGF121 from fibrin implants induces local and controlled blood vessel growth. Circulation research 2004, 94(8): Zisch AH, Schenk U, Schense JC, Sakiyama-Elbert SE, Hubbell JA. Covalently conjugated VEGF--fibrin matrices for endothelialization. Journal of controlled release : official journal of the Controlled Release Society 2001, 72(1-3): Ehrbar M, Schoenmakers R, Christen EH, Fussenegger M, Weber W. Drug-sensing hydrogels for the inducible release of biopharmaceuticals. Nature materials 2008, 7(10): Rosenbaum-Dekel Y, Fuchs A, Yakirevich E, Azriel A, Mazareb S, Resnick MB, et al. Nuclear localization of long-vegf is associated with hypoxia and tumor angiogenesis. Biochemical and biophysical research communications 2005, 332(1):