Amicon ultracel filter units

The C7 recombinant protein polymer (see Fig. S1 for full amino acid sequence) was cloned, synthesized, and purified as reported previously.[1 (link)] In brief, the DNA sequence encoding the C7 block copolymer was cloned into the pET-15b vector (Novagen) and transformed into the BL21(DE3)pLysS Escherichia coli host strain (Life Technologies). Protein was expressed following isopropyl β-D-1-thiogalactopyranoside (IPTG) induction, purified by affinity chromatography via the specific binding of N-terminal polyhistidine tag to Ni-nitrilotriacetic acid resin (Qiagen), buffer exchanged into phosphate-buffered saline (PBS), and concentrated by diafiltration across Amicon Ultracel filter units (Millipore) with 30 kDa Molecular Weight Cut-Off (MWCO). Protein identity and purity were confirmed by gel electrophoresis, MALDI-TOF mass spectrometry, and amino acid compositional analysis (data not shown).
C7 protein used in in vivo experiments was further subjected to lipopolysaccharide (LPS) removal by four cycles of phase separation and temperature transition extraction with Triton X-114. Residual Triton X-114 was removed by overnight incubation with Bio-Beads SM-2 Adsorbents (Biorad), and the PyroGene Recombinant Factor C Endotoxin Detection Assay kit (Lonza) was used to confirm the reduction of LPS levels to below 5 EU/mg in the final C7 protein solutions.

SHIELD was synthesized as previously reported (Cai et al. 2015 (link)). Briefly, 8-arm polyethylene glycol sulfone (PEG-VS) (MW 20 kDa) was purchased from JenKem Technology (Plano, TX) and the custom peptide P1 (sequence: EYPPYPPPPYPSGC) was purchased from Genscript Corp (Piscataway, NJ, USA). The PEG-P1 copolymer was synthesized by reacting 8-arm PEG-VS in excess P1 in the presence of tris(2-carboxyethyl)phosphine. Unbound P1 is removed via dialysis. The DNA encoding the C7 linear protein block copolymer was cloned into the pET-15b vector (Novagen) and transformed into the BL21(DE3)pLysS Escherichia coli host strain (Life Technologies). The protein was expressed following isopropyl β-D-1-thiogalactopyranoside induction, purified by affinity chromatography via the specific binding of N-terminal polyhistidine tag to Ni-nitrilotriacetic acid resin (Qiagen), dialyzed against PBS, and concentrated by diafiltration across Amicon Ultracel filter units (Millipore). Each of the seven WW domains in C7 was treated as one C unit, and each pendant peptide group in PEG-P1 was treated as one P unit. SHIELD was formed by 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.