Figure 3

The generation of expression and purification of target protein that was analyzed using 12% SDS-PAGE. (A) Protein molecular weight standards are shown in lane 1. Numbers on the left are the MW of the standards in kDa. Lanes 1, 2, and 3 show the controls without induction by IPTG that were 20 μL aliquots of whole cell lysates from 10 mL PGEX-6P-1-WT-GST-TMV-CP₃₂-BL ₂₁ (DE ₃ )-RIL, pET28a-His-TMV-CP₁₂-BL ₂₁ (DE ₃ )-RIL, and pET28a-TR-His-TMV-CP₁₉-BL ₂₁ (DE ₃ )-RIL cultures, respectively. Lanes 4, 5, and 6 correspond to the cultures after expression that were from 20 μL aliquots of whole cell lysates from 1 L PGEX-6P-1-WT-GST-TMV-CP₃₂-BL ₂₁ (DE ₃ )-RIL cultures with IPTG. A new protein band at 43.5 kDa corresponds to the target protein GST-TMV-CP. Lanes 7 and 8 correspond to the cultures after expression that were from 20 μL aliquots of whole cell lysates from 1 L pET28a-TR-His-TMV-CP₁₂-BL ₂₁ (DE ₃ )-RIL and pET28a-TR-His-TMV-CP₆₈-BL ₂₁ (DE ₃ )-RIL culture with IPTG, respectively. A new protein band at position 18.5 kDa corresponds to the target protein. Lanes 9 and 10 correspond to the cultures after expression that were from 20 μL aliquots of whole cell lysates from 1 L pET28a-TR-His-TMV-CP₆₂-BL ₂₁ (DE ₃ )-RIL and pET28a-TR-His-TMV-CP₁₉-BL ₂₁ (DE ₃ )-RIL cultures with IPTG, respectively. A new protein band at position 18.5 kDa corresponds to the target protein. (B) WT-GST-TMV-CP₃₂ protein is shown in lanes 1 and 2 purified using nickel-nitrilotriacetic acid (Ni-NTA) column. (C) WT-His-TMV-CP₁₂ protein is shown in Lanes 1 and 2 purified using Ni-NTA column. (D) TR-His-TMV-CP₁₉ protein is shown in Lanes 1 and 2 purified using Ni-NTA column. (E) WT-TMV-CP₃₂ protein cleaved GST-tags is shown in 12% SDS-PAGE gel filtration purified using HiLoad 16/60 Superdex 200 pg column. (F) WT-His-MV-CP₁₂ protein is shown in 12% SDS-PAGE gel filtration purified using HiLoad 16/60 Superdex 200 pg column. (G) TR-His-MV-CP₁₉ protein is shown in 12% SDS-PAGE gel filtration purified using HiLoad 16/60 Superdex 200 pg column.