SHIELD Synthesis and Purification
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Corresponding Organization : Stanford University
Variable analysis
- Mixing C7 and PEG-P1 copolymer to achieve a C:P ratio of 1:1 and a final concentration of 10% w/v in PBS
- Formation of SHIELD
- 8-arm polyethylene glycol sulfone (PEG-VS) (MW 20 kDa) purchased from JenKem Technology (Plano, TX)
- Custom peptide P1 (sequence: EYPPYPPPPYPSGC) purchased from Genscript Corp (Piscataway, NJ, USA)
- Tris(2-carboxyethyl)phosphine used for the synthesis of PEG-P1 copolymer
- Dialysis to remove unbound P1
- Cloning of DNA encoding the C7 linear protein block copolymer into the pET-15b vector (Novagen) and transformation into the BL21(DE3)pLysS Escherichia coli host strain (Life Technologies)
- Protein expression following isopropyl β-D-1-thiogalactopyranoside induction
- Purification of the protein by affinity chromatography via the specific binding of N-terminal polyhistidine tag to Ni-nitrilotriacetic acid resin (Qiagen)
- Dialysis against PBS and concentration by diafiltration across Amicon Ultracel filter units (Millipore)
- No positive or negative controls were explicitly mentioned in the input protocol.
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