Part:BBa_K3143338
INP-GS linker-SpyTag
INP is an outer membrane-bound protein from Pseudomonas syringae [1]. A SpyTag is fused to INP so that any protein fused to SpyCatcher can be fixed on cell membrane through SpyTag-SpyCatcher system. See more in csgA-SpyTag BBa_K3143338 and csgA-SpyTag-INP-SpyTag BBa_K3143339.
Figure 1: The SpyTag/SpyCatcher system
Characterization
We designed an experiment to measure the capture ability of CsgA–SpyTag,INP–SpyTag and CsgA–INP-SpyTag. We used sfGFP–SpyCatcher protein expressed and lysed from E.coli BL21 as the indicator.
We first expressed SpyCatcher-GFP protein in B Cell, and then collected the SpyCatcher-GFP protein in the supernatant by cell disruption and high-speed centrifugation. Then we added a certain amount of SpyCatcher-GFP protein to the bacterial culture medium of CsgA–SpyTag, INP–SpyTag and CsgA–INP-SpyTag, reacting for two hours.
Then, we centrifuged the reaction system and measure the fluorescence of the supernatant. The decrease of florescence indicated the amount of sfGFP – SpyCatcher protein the A cell surface captured.
As shown in the results in Fig 2a, we saw that the expression band of SpyCatcher-GFP was clearly visible on the PAGE gel. In Fig 2b, we can see that after the reaction, the fluorescence values of different groups were decreased, and CsgA–INP-SpyTag was the most significant one.
Figure 2: A PAGE Gel showed that SpyCatcher-GFP was succefully expressed and collected B Florescence measurement of csgA, INP, csgA+INP group
Reference
1. Kim D, Ku S. Bacillus cellulase molecular cloning, expression, and surface display on the outer membrane of Escherichia coli[J]. Molecules, 2018, 23(2): 503.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 405
- 1000COMPATIBLE WITH RFC[1000]
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