Difference between revisions of "Part:BBa K1159200"

Revision as of 00:42, 21 October 2019

Splitted and engineered N-terminal FbaB for isopeptide bound formation (SpyCatcher) in RFC[25]

This part codes for a protein that recognizes and forms a covalent isopeptide bound to a oligopeptide. That oligopeptide can be found under BBa_K1159201. 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[25] 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

Experimental Evidence

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).

Improvment

Inspired by the self-catalysis, Peking iGEM 2016 fused three SpyTag spaced by (VPGVG)4 with 6xHistag in N-terminal and other functional protein modules in C-terminal. The network forming capacity is not affected. Moreover, E.coli has successfully secreted our fused protein when a few of specific signal peptides are added.

Fig. 1. Exploration of the polymerization ability of the 3A-SUP/3A-mSA with 3B. Left: the Coomassie Blue staining gel of basic experiment, which illustrates the basic cross-linking ability of 3A-SUP/3A-mSA and 3B. Lane 1, Thermo® Protein Marker; lane 2, 3A-SUP; lane 3, 3A-mSA; lane 4, 3A-SUP+3A-mSA; lane5, 3B; lane 6, 3A-SUP+3B; lane 7, 3A-mSA+3B. (Molecular Weight: 3A-SUP, 21.4kDa; 3A-mSA, 25.4kDa; 3B, 55.4(62) kDa. Right: A, the OD value of the lane 5 of oligomers produced by the mix of 3A-SUP and 3B. Peaks illustrate the monomers and the possible products, ①, 3A-SUP (21.4kDa); ②, 3B (62kDa); ③, 1x 3A-SUP+1x 3B (83.4kDa); ④, 3x 3A-SUP+1x 3B (126.2kDa); ⑤, 2x 3A-SUP+2x 3B (166.8kDa); ⑥, 4x 3A-SUP+4x 3B (333.6kDa). B, the OD value of the lane 6 of oligomers produced by the mix of 3A-mSA and 3B. Peaks illustrate the monomers and the possible products, ①, 3A-mSA (25.4kDa); ②, 3B (62kDa); ③, 1x 3A-mSA+1x 3B (87.4kDa); ④, 1x 3A-mSA+2x 3B (149.4kDa); ⑤, 2x 3A-mSA+2x 3B (174.4kDa); ⑥, 3x 3A-mSA+4x 3B (324.2kDa). (The software lane 1D was used to draw the graph.) (“3A-SUP” stands for “Triple SpyTag-SUP”, “3A-mSA” for “Triple SpyTag-mSA”, and “3B” is the abbreviation of “Triple SpyCatcher”)

We found that some new bands appeared above the band of 3B when it was mixed with monomers containing A, which demonstrated that our idea of forming functional hydrogel was executable. The products were mainly oligomers (Fig. 2 A-F, Table 1), for it is easy to form loops, which hindered the linkage between different monomers at such low concentration. Interestingly, with the restriction that A is equal to B in number, and the content of 3B was constant initially, the crosslinking ability at low concentration of these monomers were different with each other by comparing the surplus content of 3B. What’s more, it was surprising that the position of 3B band (~62kDa) was not accord with its theoretical weight (55.4kDa). Further experiments will be done to understand the differences.

If you want to learn more about Peking’s polymer network and the role of SUP in this network, please click here https://parts.igem.org/Part:BBa_K1989000", https://parts.igem.org/Part:BBa_K1989001" or https://parts.igem.org/Part:BBa_K1989002".

Reference

http://www.ncbi.nlm.nih.gov/pubmed/22366317 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)

Characterization by iGEM Kyoto 2019

We measured the time development of SpyCatcher-SpyTag bond formation. An equal amount of SpyCatcher protein (SpyC) and SpyTag inserted TmEncapsulin (SpyTmEnc) were mixed and incubated at room temperature. At different time points, 10 min, 30 min, 60 min, 180 min, 360 min, 1200 min, the reaction was stopped by adding 2x SDS sample buffer. Mixed samples were assessed with SDS-PAGE. The intensities of the conjugated bands were quantified.

As shown in Fig. 1, conjugated bands become evident gradually (labeled with arrow). Signals were quantified and summarized in Fig. 2. The reaction looks saturated after 360 minutes, even though substrates still remain a lot. This might be explained by water evaporation while incubation. Otherwise, it is possible that a bound protein prevents another protein from binding to near sites on a capsule cage. If it is the case, it might limit the number of binding proteins on a capsule.

Fig. 1 Time development of SpyCatcher-Tag bond formation 3µL of SpyCatcher protein (SpyC) solution and 3µL of SpyTag inserted TmEncapsulin (SpyTmEnc) solution was mixed, then placed for several times at room temperature (10 min, 30 min, 60 min, 180 min, 360 min, 1200 min). The concentrations of protein solutions are SpyC: 500ng/µL SpyTmEnc: 1000ng/µL. The reaction was stopped by adding 2x SDS sample buffer. Mixed samples were assessed with SDS-PAGE for 30 min in 200V. CBB-stained.

Fig. 2 Quantification of conjugated band Conjugated bands’ intensity was quantified with ImageJ. Orange dots show averages value of three experiments. Blacklines show standard deviations. The time point 60min was deleted because it included negative value.

Sequence and Features