Generation and Characterization of Damaged Pfs230D1+ Proteins

Two types of damaged Pfs230D1+ proteins were generated from the reference protein [17 (link)], one was trypsin-treated Pfs230D1+ (Tryp-D1+) and the other was reduced-and-alkylated Pfs230D1+ (R&A-D1+). Tryp-D1+ was made using Immobilized Trypsin (Promega, Madison, WI, USA; Cat No V9012) following the manufacturers’ instruction. The protein concentration of Tryp-D1+ was determined by DS-11 FX+ (DeNovix, Wilmington, DE, USA). The observed concentration of Tryp-D1+ (0.45 mg/mL) was within 5% error from a theoretical concentration (0.47 mg/mL) calculated from the original amount of protein used for the reaction and the final volume of solution. To generate R&A-D1+, 1.49 mL of 1M DDT was mixed with 1 mg of original Pfs230D1+ protein for 30 min at 60 °C, then 41 µL of 1 M IAA was added and incubated for 30 min at 37 °C. The reaction was stopped by adding 0.58 mL of BME. Using Slide-A-Lyzer Dialysis Cassettes (3.5 kDa) and 3.0 kDa Spin Concentrators, the R&A-D1+ sample was buffer exchanged to TBS (pH8.0) and concentrated to the same protein concentration of the original Pfs230D1+ protein (0.67 mg/mL). The protein concentration in the final product was determined by DS-11 FX+.