Part:BBa_K4645003
Fusion protein formed by CsgA and Hsa with (GGGGS)4 as linker.
The NR2 domain of Hsa is the sialic-binding core of Hsa protein, and CsgA is the extracellular fiber component of Escherichia coli biofilm formation. The two proteins were connected with a flexible linker to enable them to colonize on cat teeth and form a stable biofilm, creating conditions for the realization of subsequent neutralization.A 6x His tag was added to the C-terminus of the protein for purification or specificity testing in experiments.
Usage and Biology
Curli mediate host cell adhesion and invasion and play a critical role in biofilm formation. Curli filaments consist of CsgA, the major subunit, and CsgB, the minor subunit.CsgA proteins can self-assemble into amyloid nanofibers, characterized by their hierarchical structures across multiple length scales, outstanding strength and their structural robustness under harsh environments.The extracellular stacking of this protein forms the scaffold of the E. coli biofilm.Many streptococci adhere to protein-attached carbohydrates expressed on cell surfaces using Siglec-like binding regions (SLBRs).SLBRs are usually found within the context of serine-rich repeat proteins, which form fibrils extending from the bacterial surface. The Siglec domain contains a ΦTRX sequence motif15 that recognizes Neu5Acα2-3Gal in the context of larger glycans[1].
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 37
Illegal PstI site found at 861 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 37
Illegal PstI site found at 861 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 37
Illegal PstI site found at 861 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 37
Illegal PstI site found at 861 - 1000COMPATIBLE WITH RFC[1000]
Functional Parameters
To obtain the CsgA-Hsa protein, we constructed pET-28a(+)-CsgA-Linker-Hsa and transferred it to E. coli BL21(DE3) (Figure 1). The E. coli strain was cultured to OD600=0.6, induced with 1 mM IPTG, and alllowed the bacterial to grow 16h at 16℃, 160rpm. CsgA-Hsa's molecular weight is about 43kDa. The fusion protein CsgA-Hsar was confirmed to be expressed by SDS-PAGE analysis (SDS-PAGE Preparation kit , Sangon Biotech (Shanghai) Co., Ltd), although the linker of the fusion protein may have been broken during sonication.
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