Quickchange site directed mutagenesis protocol

Recombinant protein expression used a pET21a(+) expression vector (EMD Millipore, Billerica, MA) with a codon-optimized synthetic gene encoding the 140 amino acid “mature” form of human WT FGF-1 and with an N-terminal 6× His tag. The QuickChange^™ site-directed mutagenesis protocol (Agilent Technologies, Santa Clara, CA) was used to introduce all FGF mutations and were confirmed by DNA sequencing (Biomolecular Analysis Synthesis and Sequencing Laboratory, Florida State University). Recombinant WT FGF-1 protein was expressed from pET21a(+)/BL21(DE3) Escherichia coli as previously described.^{31 (link)} Recombinant disulfide mutants were expressed from SHuffle T7 Express E coli (New England BioLabs, Ipswich, MA) and Luria broth media. The E coli culture was incubated at 30°C until OD₆₀₀ = 0.6, at which point the temperature was shifted to 20°C and 1 mM isopropyl-β-D-thio-galactoside was added to induce protein expression with overnight incubation. The expressed protein was purified using sequential column chromatography on Ni-nitrilotriacetic acid affinity resin (Qiagen, Valencia, CA) and heparin Sepharose resin (GE Life Sciences, Pittsburgh, PA). Protein purity was evaluated by gel densitometry of Coomassie blue–stained SDS-PAGE. An extinction coefficient of E_280nm (0.1 %, 1 cm) = 1.26^{31 (link)} was used for concentration determination of WT FGF-1 and all mutant proteins.

Primers were designed using the QuickChange Site-Directed Mutagenesis Protocol (Agilent) and are listed in Supplementary Materials and Methods. PCR was performed using iProof DNA polymerase and GC buffer using primers specific for each mutation (Bio-Rad). Mutations were confirmed by Sanger sequencing using vector-specific primers.
Aside from the mutagenesis screens, all experiments were repeated at least three times, with representative graphs or blots shown. Error bars represent SDs.