Recombinant protein production

The BBA66 coding sequence minus the signal sequence and lipidation motif was amplified by PCR from B. burgdorferi B31 using primers designed for expression cloning into the pETite N-His vector according to the T7 Expresso system instructions (Lucigen, Middleton, WI) [32]. The constructed expression plasmids were transformed into E. coli 10G (Lucigen) and selected for growth on LB plates supplemented with 50 μg/ml kanamycin. DNA sequencing of the plasmid insert from transformants was performed to confirm the BBA66 coding sequence was correctly in frame for expression. Recombinant BBA66 pET-plasmids were purified by miniprep (Qiagen, Valencia, CA) and transformed into E. coli BL21(DE3) (Lucigen). Following transformant screening for the appropriate clones, colonies were grown in LB-kanamycin (50 μg/ml) broth, and recombinant protein expression was induced by the addition of IPTG (isopropyl-D-thiogalactopyranoside; 1 mM). Cells were harvested at late-log-phase growth, and recombinant protein was purified under nondenaturing conditions using a nickel-nitrilotriacetic acid (Ni-NTA) Fast Start His Tag affinity purification kit (Qiagen). Proteins were dialyzed into phosphate buffered saline (PBS, pH 8.0) and quantified by bicinchoninic acid (BCA) assay (Thermo-Fisher Scientific, Rockford, IL). Recombinant OspC was generated in same manner as described previously [32].

FH2/Daam1 recombinant protein from the Y2H gene fragment clone was produced and purified by the T7 Expresso system in the pETite N-His vector as described above. The fragment was PCR-amplified from the Y2H prey plasmid.