Part:BBa_K3089022

rBalcp19K for yeast expression

This composite part is meant to express csgA-linker-mfp5-linker-mfp3 fusion genes under T7 promoter, and 7XHis-tag was fused on the C terminal to achieve affinity protein purification. It is a updated version from T7 promoter+csgA-linker-mfp5-His (BBa_K1583021) and T7 promoter+csgA-linker-mfp5-mfp5-His (BBa_K1583023). 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. CsgA is an amyloid-like protein encoded on genome of E.coli MG1655 providing cohesive mechanical strength. 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).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 314
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 314
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 314
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 314
  
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Three different experiments were done to characterise the BBa_K3089022 biobrick:

• protein expression
• protein purification

Protein expression

Figure 2. The circuit of the protein BBa_K30889022

csgA-linker-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 the expression, whole cells were collected after induction by centrifuging and prepared for SDS-PAGE. Results showed that no obvious protein bands of CsgA-mfp5-mfp3(~30 kDa) could be observed on lane WC compared with lane pET28b (pET28b empty vector), which means the expression of this protein is not well in BL21(DE3) Rosetta.

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.

Protein purification

For we make producing underwater bio-adhesives as the final goal of our project, though no obvious protein bonds of interest could be observed, we straightly went on protein purification. CsgA is an amyloid-like protein characterised by β-strands, and CsgA monomers would form aggregates after expression inside cells. Therefore, denature protein purification methods were used — weak bands presented on the lane E1. The mixed solutions were then loaded on the columns and dialysed with PBS buffer (PH=6.0) to wash away imidazole. Meanwhile, the protein was concentrated. After that, the concentrated protein was put under 4℃ for 72 hours to make it renature. Protein concentrations of CsgA-linker-mfp5-linker-mfp3 were measured by BCA assay, and its yield is 0.7mg/L.

Figure 4. Coomassie-stained SDS-PAGE gels confirm purification of the expressed protein Csg-linker-mfp5-linker-mfp3 by cobalt-resin columns. Lanes: M, protein molecular weight marker; WC, whole-cell sample of recombinant proteins; E, eluted proteins. 12% SDS-PAGE gels were used for the analyses.

Reference

Zhong, C. et al., 2014. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nature nanotechnology, 9(10), pp.858–66