Genetailor

Two point mutations that change amino acid 85 from a Methionine to either a Glycine (M85G) or Lysine (M85K) were introduced into the pcDNA-Elafin vector sequence using GeneTailor® (Invitrogen, Carlsbad, CA) and transfected into OVCAR8. Mutations were confirmed by DNA sequencing. Stable cell lines were constructed as described above.

Site-directed mutants of EcPutA were made from the full-length construct of wild-type EcPutA (UniProt Accession ID:P09546) (EcPutA-pET14b) using Stratagene QuikChange or Invitrogen GeneTailor mutagenesis kits. The primers used for mutagenesis were purchased from Integrated DNA technologies and are listed in Table S1 (see Supporting Information). C-terminal deletion mutants were generated by inserting a stop codon immediately after the codons for residues 1308, 1314, and 1317. All of the EcPutA mutants were confirmed by DNA sequencing. EcPutA wild-type and mutant proteins were overexpressed in E. coli strain BL21(DE3) pLysS and purified by Ni-NTA Superflow affinity (Qiagen) chromatography using a N-terminal 6xHis-tag as previously described.^{50 (link),53 (link)} The N-terminal His-tag was retained for EcPutA wild-type and mutants in subsequent experiments. Purified proteins were stored in 50 mM Tris buffer containing 50 mM NaCl, 0.5 mM EDTA, 0.5 mM Tris (3-hydroxypropyl) phosphine (THP), and 10% glycerol (pH 7.5).