Designed by: Dong-Jiunn Jeffery TRUONG Group: iGEM13_TU-Munich (2013-09-14)
Splitted and engineered C-terminal FbaB for isopeptide bound formation (SpyTag) in RFC
This part codes for a oligopeptide that is recognized and bound by a covalent isopeptide bound to a protein, called SpyCatcher. That catcher protein for this oligopeptdie can be found under BBa_K1159200. This part is flanked by RFC pre- and suffix for further protein fusions.
This part codes for a protein that recognizes and forms a covalent isopeptide bound to a oligopeptide. That oligopeptide can be found under BBa_K1159200. In contrast to the original SpyCatcher this part only contains the essential part for forming isopeptide bounds and does not contain a N-terminal His-tag and TEV cleavage site. Furthermore two amino acids were exchanged to improve the function, Ile34 was replaced by Glu34 which enhances the reaction rate, also the exposed hydrophobic redidue Met69 was replaced by Tyr69. This part is flanked by RFC pre- and suffix for further protein fusions.
Figure 1: Schematic of the working mechanism of post-translational non-canonical protein fusion using the SpyTag/SpyCatcher system
Proper funtion of the construct was verified in a pulldown assay using 10 μM of SpyCatcher with N-terminal HisTag which was incubated with 5 μM of C-terminal spytagged SYFP2 for 1 h in PBS. Afterwards HisTag beads were added to bind the fusion product, resulting in a decrease of spytagged SYFP2 in the supernatant. The results of this assay are shown in Figure 2. The results show that after one hour at room temeperature, a two fold change in relative absorbance was observed. This verifies that the construct is working as intended.
Figure 2: Experimental setup of the pulldown assay for the SpyTag/SpyCatcher system.
Figure 3: Results of the pulldown assay for the SpyTag/SpyCatcher system.
Furthermore the two interaction partners were mixed, incubated and analyzed via SDS Page (reducing conditions) and coomassie staining.
Figure 4: Results of the SDS page of SpyTag/SpyCatcher system products (reducing conditions).
[Zakeri et al., 2012] Zakeri B, Fierer JO, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M. (2012). Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A. 20;109(12)
Nucleotide sequence in RFC 25, so ATGGCCGGC and ACCGGT were added (in italics) to the 5' and 3' ends: (underlined part encodes the protein) ATGGCCGGCGCTCATATT ... CCAACTAAGACCGGT ORF from nucleotide position -8 to 45 (excluding stop-codon)
Amino acid sequence: (RFC 25 scars in shown in bold, other sequence features underlined; both given below)