1Supplementary Materials:
Figure 1supplemental: Schematic diagram of cloning steps for construction of p2.2-Nb. VEGFR2-specific nanobody coding sequence (3VGR19) was PCR amplified with primers containing Eco91I recognition sites using pHEN6C-3VGR19 as template. PCR product and 2.2 plasmid were digested with Eco91I restriction enzyme and after purification were ligated to produce the p2.2-Nb vector. The final construct (p2.2-Nb) was confirmed by restriction digestion analysis and DNA sequencing. E1 is fusogenic protein of sindbis virus and E2 is the protein responsible for attachment (binding) of sindbis virus to its cognate receptors. E3 and 6K are leader sequences for E2 and E1, respectively; ZZ denotes the sequences encoding the IgG binding domain of protein A from S. aureus. CMV stands for Human cytomegalovirus(CMV) immediateearly promoter. AmpR is Ampicillin resistance gene. His indicates His-Tag (Histidine tag or HHHHHH).
Figure 2supplemental:Schematic diagram of cloning steps for construction of p2.2-VEGF121. The coding sequence of VEGF121 (a VEGF sequence which encodes an isoform of 121 amino acid) was PCR amplified with primers containing Eco91I recognition sites using pET26-VEGF121 as template. PCR product and 2.2 plasmid were digested with Eco91I restriction enzyme and after purification were ligated to produce the p2.2-VEGF121 vector. The final construct (p2.2-VEGF121) was confirmed by restriction digestion analysis and DNA sequencing. E1 is fusogenic protein of sindbis virus and E2 is the protein responsible for attachment (binding) of sindbis virus to its cognate receptors. E3 and 6K are leader sequences for E2 and E1, respectively; ZZ denotes the sequences encoding the IgG binding domain of protein A from S. aureus. CMV stands for Human cytomegalovirus(CMV) immediateearly promoter. AmpR and KanR are Ampicillin and Kanamycin resistance genes respectively.
Figure 3supplemental:Schematic diagram of cloning steps for construction of p2.2-L. The Sequence corresponding to the linker encoding HA-tag, G4S and His-tag was synthesized by Biomatik in the context of pUC57 plasmid flanked by Eco91I recognition sites (pUC57-Linker). Both pUC57-Linker and 2.2 plasmid were digested by Eco91I. Subsequently gel purified linker fragment was cloned into 2.2 plasmid to provide the final p2.2-L plasmid as control vector in this study. E1 is fusogenic protein of sindbis virus and E2 is the protein responsible for attachment (binding) of sindbis virus to its cognate receptors. E3 and 6K are leader sequences for E2 and E1, respectively; ZZ denotes the sequences encoding the IgG binding domain of protein A from S. aureus. CMV stands for Human cytomegalovirus(CMV) immediateearly promoter. AmpR is Ampicillin resistance gene.
Figure 4supplemental:Energy minimization of cSVE2s. a) 2.2-Nb and b) 2.2-VEGF121.As depicted in the graphs, the modeled 3D structures were energy minimized during 6000 psusing steep descent algorithm.
Figure 5supplemental:Pressure (a) and temperature (b) equilibration of cSVE2s. The temperature and pressure of systems were coupled at 300 °K and 1 bar using V-rescale and Parrinello-Rahman algorithms, respectively.
Figure 6supplemental:Quality assessment of the refined structures.(a) E2-Nb and (b) E2-VEGF121. More than 93 %of residues are located in the allowed regions for both fused proteins.
Figure 7 supplemental: Productiontiters of unconcentrated lentiviral vectors pseudotyped with VSV-G, 2.2-Nb and 2.2-VEGF121 determined by P24 ELISA. To produce LVs pseudotyped with targeted cSVE2s (2.2-Nb, 2.2-VEGF121) or non-targeted positive control of transduction (VSV-G), 293T cells were transfected with 3 µg lentiviral transfer vector pLOX-CWgfp (a LV transfer vector encoding GFP as a reporter gene, 2 µg psPAX2 (packaging plasmid) and 1 µg of p2.2-Nb, p2.2-VEGF121 or pMD2.G respectively. Average production titer of LVs pseudotyped with 2.2-Nb and 2.2-VEGF121 were 66.5% and 70% ofLVs bearing VSV-G, which is regarded the standard for comparison.
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