protein expression  (Bio-Rad)

Journal: RSC Advances

Article Title: Plastic-binding peptides as anchors for protein scaffolds on synthetic plastics: opportunities and challenges

doi: 10.1039/d5ra06185g

Figure Lengend Snippet: Purification and assembly of a synthetic cohesin–dockerin scaffold complex. (A) The molecular design of all fusion proteins used for the development of the plastic-targeting complex. A plastic-targeting complex was designed using three cohesins from cellulosomal proteins (CipC, CipA, OlpB) and fused to LCIM3 on both termini. A control scaffold lacking LCIM3 was also constructed. Dockerins from Cel5A, Cel48S, and CipA were fused to distinct fluorophores via native linkers. Scaffold constructs carried an N-terminal Strep II tag; dockerin fusions had a C-terminal 6×His tag. (B) Assembly of the dockerin–cohesin complex was validated by pull-down experiments targeting the scaffold backbone using MagStrep® Strep-Tactin® XT beads. Lanes to the left of the dotted line show the purified protein fusions used for complex assembly. Lanes to the right of the dotted line represent pull-down results. The first four lanes on the right demonstrate that only the scaffold binds to the Strep-Tactin XT beads when each protein is incubated individually. When all four proteins are incubated together, the fluorophore–dockerin fusions co-elute with the scaffold (elute lane) in the Strep-Tactin XT pull-down.

Article Snippet: Strep-Tactin®XT 4Flow® resin (2-5010-025) for strep affinity protein purification was purchased from IBA lifesciences.

Techniques: Purification, Control, Construct, Incubation