Recombinant PTD-BMP2 was synthesized as previously described.11 (link)
The PTD-fusion BMP2 polypeptide was amplified using pRSET (Invitrogen, Waltham, MA, USA), a bacterial expression vector containing an Xpress epitope and a His tag. The precursor cDNA of BMP2 was amplified from the cDNA of Saos-2 osteosarcoma cells using polymerase chain reaction. The TAT sequences (RKKRRQRRR) for the PTD domain were inserted adjacent to the epitope, and the precursor cDNAs of BMP2 were cloned into a TAT-expression cassette. Following the transformation of BL21 competent cells and IPTG induction, inclusion bodies were obtained from the insoluble fraction using 1% Triton X-100 buffer. The insoluble fraction was solubilized with an 8 M urea solution, and the recombinant protein was purified using Ni-Ti beads and imidazole elution. Buffer shock was then employed to imbue high surface energy (∆G) properties. Denatured polypeptides were micellized with filtered 0.1% egg lecithin (BOC Sciences, Shirley, NY, USA) via bath sonication.28 (link),29 (link)
The PTD-fusion BMP2 polypeptide was amplified using pRSET (Invitrogen, Waltham, MA, USA), a bacterial expression vector containing an Xpress epitope and a His tag. The precursor cDNA of BMP2 was amplified from the cDNA of Saos-2 osteosarcoma cells using polymerase chain reaction. The TAT sequences (RKKRRQRRR) for the PTD domain were inserted adjacent to the epitope, and the precursor cDNAs of BMP2 were cloned into a TAT-expression cassette. Following the transformation of BL21 competent cells and IPTG induction, inclusion bodies were obtained from the insoluble fraction using 1% Triton X-100 buffer. The insoluble fraction was solubilized with an 8 M urea solution, and the recombinant protein was purified using Ni-Ti beads and imidazole elution. Buffer shock was then employed to imbue high surface energy (∆G) properties. Denatured polypeptides were micellized with filtered 0.1% egg lecithin (BOC Sciences, Shirley, NY, USA) via bath sonication.28 (link),29 (link)
