Part:BBa_K3089017

rBalcp19K-linker-mfp5-His fusion protein

This composite part is meant to express rBalcp19k-linker-mfp5 fusion genes under the T7 promoter, and 7XHis-tag was fused on the C terminal to achieve affinity protein purification. It is an updated version from the basic part rBalcp19k (<a href="https://parts.igem.org/Part:BBa_K3089008"target="_blank">BBa_K3089008</a>)which is expressed in Pichia pastoris as well.

Mfp5 is the mussel foot protein from Mytilus galloprovincialis responsible for interface adhesion. rBalcp19k, the barnacle adhesive protein from Balanus albicostatus, its homologous gene serves as adhesive proteins, also playing the role of interfacial adhesion in a way that mfp5 does in mussels(Figure 1). We linked them together to achieve better adhesive ability because rBalcp19k can self-assemble into aggregated nanofibers at acidic pHs.

This composite part would be a promising new generation of bio-inspired adhesives for a wide range of applications. The sequence of this composite part is optimised to achieve better expression in Pichia pastoris.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 274
  Illegal PstI site found at 23
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 274
  Illegal PstI site found at 23
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 274
  Illegal XhoI site found at 244
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 274
  Illegal PstI site found at 23
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 274
  Illegal PstI site found at 23
  
• 1000
  COMPATIBLE WITH RFC[1000]

Figure 1. Protein distribution in barnacle cements

Characterization

Three different experiments were done to characterise the BBa_K3089017 bio-brick:

• Molecular cloning
• Protein purification

Molecular cloning

Figure 2. Illustration of gene insertion into Pichia pastoris GS115 genome. Box in blue indicates the recombinant protein CsgA-linker-mfp5-mfp5.

CsgA-linker-mfp5-mfp5-His was synthesised and cloned to yeast Pichia pastoris, and one strain was verified by gel electrophoresis and sequencing (Figure 2).

Figure 3. Determination of gene insertion into the Pichia genome by gel electrophoresis.

Protein expression

Proteins were expressed in small scale induced by methanol. 4 strains of rBalcp19k-mfp5 demonstrated clear bands around 25kDa on Lane S (culture medium) after expressing them for 48 hours. The predicted size of rBalcp19k-mfp5 is 28kDa, larger than shown in the gel(Figure 3). This may be due to the acidification of the culture medium after long-time incubation.

Figure 4. SDS-PAGE confirm expression(30℃，48h) of CsgA-mfp5-mfp5 and rBalcp19k-mfp5(4 colonies) expression. P, cell pellets. S, supernatant.