Part:BBa_K3089025

T7 promoter+mfp5-linker-mfp3-His

This composite part is meant to express mfp5-linker-mfp3-His fusion genes under the T7 promoter, and 7XHis-tag was fused on the C terminal to achieve affinity protein purification. Compared to BBa_K3089022, this part does not contain CsgA, which is responsible for cohesion. Linking mfp5-mfp3 together to provide better adhesion mimics the natural distribution of Mfp5 and Mfp3 proteins in Mussel feet (Figure 1), which may give better performance in making underwater bioadhesives then mfp5 alone and mfp5-mfp5. Mfp5 and Mfp3 are mussel foot proteins from Mytilus galloprovincialis responsible for interface adhesion.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 47
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 159
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 47
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 47
  
• 1000
  COMPATIBLE WITH RFC[1000]

This composite part is meant to express mfp5-linker-mfp3-His fusion genes under the T7 promoter, and 7XHis-tag was fused on the C terminal to achieve affinity protein purification. Compared to BBa_K3089022, this part does not contain CsgA, which is responsible for cohesion. Linking mfp5-mfp3 together to provide better adhesion mimics the natural distribution of Mfp5 and Mfp3 proteins in Mussel feet (Figure 1), which may give better performance in making underwater bioadhesives then mfp5 alone and mfp5-mfp5. Mfp5 and Mfp3 are mussel foot proteins from Mytilus galloprovincialis responsible for interface adhesion.

Figure 1. Schematic view of mussel foot proteins (mfps) in a byssal plaque of Mytilus showing the approximate location of each of the major proteins (Lee, Messersmith et al. 2011).

Characterization

Three different experiments were done to characterise the BBa_K3089025 bio-brick:

• protein expression

Protein expression

Figure 2. The circuit of the protein BBa_K30889025

The predicted size of Mfp5-linker-mfp3 is 16.17 kDa and the isoelectric point is 10.41. Mfp5-linker-mfp3 was cloned into pET28b and expressed in E.coli BL21(DE3) Rosetta by 500μM IPTG for 5h at 37℃. In order to detect its expression, whole-cells were collected after induction by centrifuging and prepared for SDS-PAGE. Results showed that obvious protein bands of (~20 kDa) could be observed on lane WC compared with lane pET28b (pET28b empty vector)(Figure 1A), which means the expression of this protein could be detected in BL21(DE3) Rosetta. Quantitative densitometry analysis of SDS-PAGE indicated that Mfp5-linker-mfp3 expressed better than Mfp5 alone or CsgA and rBalcp19k related fusion proteins under the same expression conditions (Figure 1B).

Figure 3. Detection of the expression level of all recombinant proteins by SDS-PAGE. (A) SDS-PAGE of whole-cell lysates of all recombinant proteins. Red arrows show the predicted place of certain proteins. (B) Protein SDS-PAGE bands optical densities were measured by quantitative densitometry of SDS-PAGE of whole-cell aliquots.

Reference

Lee, B. P., P. B. Messersmith, J. N. Israelachvili and J. H. Waite (2011). "Mussel-Inspired Adhesives and Coatings." Annu Rev Mater Res 41: 99-132.