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Figure 2 | BMC Biotechnology

Figure 2

From: Biotinylated-sortase self-cleavage purification (BISOP) method for cell-free produced proteins

Figure 2

Preparation of proteins by the biotinylated-sortase self-cleavage purification (BISOP) method. A. Scheme for creating the pEU-BISOP-LPETG-gene plasmids using the Gateway recombination method. The LPETG-fused target gene containing entry clone plasmid was recombined with the pEU-BISOP-GW vector by using the Gateway LR reaction. B. CBB-stained SDS-PAGE comparing the His- or biotin-tagged GFP protein (indicated using an asterisk) purified manually or by using an automated robot. Lane M: Protein MW standards. C. Biotinylation of GFP, Pfs25 (S25) and CSK proteins synthesized by the cell-free system with vectors having a single (S) or a double (D) biotin ligation site. Biotinylated proteins were detected by labeling the separated protein bands with streaptavidin-Alexa488 as described in the text, followed by scanning using the Typhoon Imager. D. CBB-stained eluted (left panel) and resin-bound (right panel) proteins. Arrow indicates the cleaved biotinylated srtA protease left on the resin. Samples (5 or 10 μL in left or right panel respectively) were loaded on the gel. E. Left panel: Biotinylated proteins were detected by streaptavidin-Alexa488; Right panel: CBB-stained proteins purified by the BISOP method. Samples (5 μL) were loaded on the gel. F. Amino acid sequences from the N-terminal end of GFP purified by the BISOP method were determined by using an amino acid sequencer (asterisks, upper panel). Rounds indicate the number of Edman degradation cycle. Underlined amino acid sequence in the lower panel shows the determined sequence. Arrowhead indicates the site cleaved by the sortase enzyme.

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