Difference between revisions of "Part:BBa K3089008"
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Revision as of 13:06, 21 October 2019


rBalcp19K

One of our adhesives. Barnacle cement proteins are very promising in making biomedical bioglues. rBalcp19K had the properties of both self-assembly and adhesion.

This composite part is meant to express rBalcp19k gene under T7 promoter. This part is also expressed in Pichia pastoris (BBa_K3089016) after codon optimization. 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(Liang, et al. 2015) (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.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 325
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 325
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 441
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 325
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 325
    Illegal AgeI site found at 304
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

Three different experiments were done to characterise the BBa_K3089008 biobrick:

  • protein expression
  • protein purification
  • Surface coating analysis

Protein expression

rBalcp19k 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 band of rBalcp19k(~19KDA) could be observed on lane WC compared with lane pET28b (pET28b empty vector), which means the expression of this protein is well in BL21(DE3) Rosetta.

Figure 2. 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

We expressed it in E.coli BL21(DE3) Rosetta, and we found bands of rBalcp19K appeared between 15kDa and 25kDa on 12% SDS-PAGE gel (Figure 5A), which meant it was successfully expressed and purified under native condition. In SDS-PAGE of rBalcp19k, we found various unexpected bands, even if we used higher concentration imidazole as washing buffer, we still couldn’t get rid of them. It seemed like they were polymers of rBalcp19k caused by self-assembly. The final yield was 2mg/L.

Figure 3A. SDS-PAGE of purified rBalcp19k (A) by affinity chromatography under native conditions. Lanes: M, protein molecular weight marker; NC, whole-cell sample of pET28b empty vector; WC, whole-cell sample of recombinant protein rBalcp19K; S, soluble cell fraction; W1, fraction.

Surface coating analysis

After obtaining a small number of recombinant proteins, surface coating analysis (see methods), the qualitative assessment of the surface adsorption ability of recombinant proteins, was performed on 2 of the most commonly used bio-related surfaces: hydrophilic glass slides and hydrophobic polystyrene tissue culture plates. As shown in Figure 3, rBalcp19K exhibited lower surface absorption abilities than rBalcp19k-mfp5, whereas almost all absorbed BSAs were washed away. Basing on our current understandings of Mfp5, we were able to predict the adhesiveness of this protein, especially when it is combined with different proteins, making it more adaptable in many environments.

Figure 4. Surface coating analysis of recombinant proteins on hydrophilic glass slides (left) and hydrophobic polystyrene (PS) plates (right).

Reference

Liang, C., et al.(2015). Protein Aggregation Formed by Recombinant cp19k Homologue of Balanus albicostatus Combined with an 18 kDa N-Terminus Encoded by pET-32a(+) Plasmid Having Adhesion Strength Comparable to Several Commercial Glues. PLoS One 10(8):e0136493.