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. 2022 Sep 3;25(10):105054. doi: 10.1016/j.isci.2022.105054

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© 2022 The Authors

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Recombinant GF production

Scale up of protein expressions for (A) FGF2 AND FGF1 cloned in pMCSG53 vector with N-terminal His6x tag and expressed in BL21(DE3) Gold cells. Targets include F1 (FGF2_Atlantic salmon); F2 (FGF2_Pufferfish); F3 (FGF1_Sheep); F4 (FGF1_Bovine) (B) PDGF-BB expressed in SHuffle T7 express cells. Target shown is P1 (PDGF-BB_Cormorant) (C) IGF1/IGF2 cloned in pMCSG53-His6x-DsbC/pMCSG53-His6x-SUMO and expressed in SHuffle T7 express cells. Targets include (K1) IGF1_Bovine (SUMO-His6x tag); (K2) IGF1_Bovine (DsbC-His6x tag); (K3) IGF1_Goose; (K4) IGF1_Frog; (J1) IGF2_Human; (J2) IGF2_Bovine; (J3) IGF2_Nile tilapia (D) TGFβ-1 cloned in pMCSG53-His6x-DsbC and expressed in SHuffle T7 express cells. Targets shown are TGFβ1_human (T1); TGFβ−1_bovine (T2); TGFβ−1_chicken (T3); TGFβ−1_little egret (T4). UC = uncut before TEV digest;C = 48h post-TEV digest; TEV protease runs at 25 kDa (marked with ˆ). After the TEV digest and a second Ni-NTA affinity chromatography step, the concentrated, purified FGF2/FGF1 runs at 15 kDa on an SDS-PAGE (marked with ★) shown in (A). PDGF-BB runs at 15 kDa corresponding to the monomer (marked with ) shown in (B). DsbC fusion IGF1/IGF2 runs at 35 kDa (marked with ∗). IGF1-SUMO runs at 20 kDa (marked with ∗∗), as seen in (C). DsbC-TGFβ-1 runs at 40 kDa (marked with #).

Inline graphic — Recombinant GF production

Scale up of protein expressions for (A) FGF2 AND FGF1 cloned in pMCSG53 vector with N-terminal His6x tag and expressed in BL21(DE3) Gold cells. Targets include F1 (FGF2_Atlantic salmon); F2 (FGF2_Pufferfish); F3 (FGF1_Sheep); F4 (FGF1_Bovine) (B) PDGF-BB expressed in SHuffle T7 express cells. Target shown is P1 (PDGF-BB_Cormorant) (C) IGF1/IGF2 cloned in pMCSG53-His6x-DsbC/pMCSG53-His6x-SUMO and expressed in SHuffle T7 express cells. Targets include (K1) IGF1_Bovine (SUMO-His6x tag); (K2) IGF1_Bovine (DsbC-His6x tag); (K3) IGF1_Goose; (K4) IGF1_Frog; (J1) IGF2_Human; (J2) IGF2_Bovine; (J3) IGF2_Nile tilapia (D) TGFβ-1 cloned in pMCSG53-His6x-DsbC and expressed in SHuffle T7 express cells. Targets shown are TGFβ1_human (T1); TGFβ−1_bovine (T2); TGFβ−1_chicken (T3); TGFβ−1_little egret (T4). UC = uncut before TEV digest;C = 48h post-TEV digest; TEV protease runs at 25 kDa (marked with ˆ). After the TEV digest and a second Ni-NTA affinity chromatography step, the concentrated, purified FGF2/FGF1 runs at 15 kDa on an SDS-PAGE (marked with ★) shown in (A). PDGF-BB runs at 15 kDa corresponding to the monomer (marked with ) shown in (B). DsbC fusion IGF1/IGF2 runs at 35 kDa (marked with ∗). IGF1-SUMO runs at 20 kDa (marked with ∗∗), as seen in (C). DsbC-TGFβ-1 runs at 40 kDa (marked with #).