Part:BBa_K5374010

SPARC/OD-EGFP. A collagen-binding domain involved in tissue repair and bone mineralization, fused wi

SPARC, containing the Osteonectin Domain (OD), interacts with the extracellular matrix to promote collagen binding and matrix remodeling. The fusion with EGFP allows for the assessment of the domain’s influence on EGFP activity, providing insight into how it may affect downstream protein functionality. This system helps screen potential tags for cell factor release in tissue engineering.

The fluorescence data were obtained by expressing various EGFP fusion proteins in E. coli, diluting each culture to an equal cell density. Fluorescence intensity was measured for each group, and the fluorescence ratio relative to EGFP (used as a standard) was calculated. The figure shows these normalized fluorescence values for each fusion protein.

Figure 1.1 The fluorescence activity of each fusion protein

The fluorescence activity of each fusion protein was tested to evaluate whether the CBDs affected EGFP activity. The relative fluorescence values of the eight CBD-EGFP fusion proteins were measured to assess the impact of the collagen-binding domains (CBDs) on EGFP activity. As shown in the graph:

• SPARC/OD-EGFP, CBD MMPs-EGFP, DB-EGFP, VWF-A Domain-EGFP, and FTD-EGFP exhibited fluorescence values close to or higher than that of the control EGFP, indicating that these CBDs did not significantly affect the bioactivity of EGFP.
• DDR-EGFP, AGR-EGFP and COMP-EGFP showed slightly lower fluorescence values, suggesting some impact on EGFP activity, but still maintaining measurable fluorescence.

Overall, the majority of CBDs retained sufficient EGFP activity, which confirms that they can be used in further experiments for controlled release without significantly impairing the functionality of the fusion proteins. These results support the selection of CBDs that balance collagen binding with maintaining protein bioactivity.

Binding affinity tests (via microscale thermophoresis) were also conducted to determine which CBDs had high binding affinity (for BMP-4) and low binding affinity (for VEGF). Based on the binding experiment shown in the graph, the results illustrate the relative binding affinities of different CBD-EGFP fusion proteins to collagen, with the binding affinity represented by the dissociation constant (Kd). The midpoint of each curve on the x-axis corresponds to the Kd value, indicating how tightly the CBDs bind to collagen.

Figure 1.2 Binding energy of each fusion protein

• FTD-EGFP shows the strongest binding to collagen, with the lowest Kd value. From the graph, the midpoint of the FTD curve is approximately at 8.3*10^-7 M, indicating a high binding affinity.
• CBD MMPs-EGFP exhibits the weakest binding to collagen, with the highest Kd value. The midpoint of the CBD MMPs curve is approximately at 6.7*10^-6 M, indicating a lower binding affinity compared to the other CBDs.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 484
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 484
  Illegal NheI site found at 922
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 484
  Illegal BglII site found at 897
  Illegal BamHI site found at 288
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 484
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 484
  Illegal NgoMIV site found at 859
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1565