Part:BBa_K4247014

This part is the N terminus of the aneroin protein (composite part BBa_K4247017) and contains a TrpL motif (MLAIFVLK) followed by an enterokinase cleavage site (DDDDK) to boost expression. The sequence of aneroin has multiple repetitions of the motif GPGNTGYPGQ (with seldom variations) and thus, was difficult to synthesise due to extreme repetitiveness. As part of our collection, Aneroin_Nt (BBa_K4247014) can be linked to Aneroin_middle (BBa_K4247015) and Aneroin_Ct (BBa_K4247016) to form the composite part Aneroin (BBa_K247017). Aneroin contains some of the typical motifs of collagens and spider silk, it can be therefore spun into a fibre.

Usage and Biology

Silk proteins are becoming increasingly popular fibres for biomaterials in many biotechnological fields, from medicine to common consumables. In 2013, Yang and colleagues discovered a new silk-like protein in the sea anemone Nematostella vectensis. This sea-star anemone lives along the coasts of England and the USA, and has been used for a long time as a model organism for the phylum Cnidaria. The protein was an hypothetical protein found in the model genome when Yang et al. (2013) discovered it. It’s characterised by its localisation in the nematocysts of the tentacles (the harpoon-like weapons of Cnidarians) and by the repetitive motif GPGNTGYPGQ.

The protein has high levels of glycine and proline, the GXX repeat motif of collagens and the GPGXX repeat of spider silks, suggesting its involvement with prey capture and potential for fiber formation. Considerinng its properties, we exploited it to include it in a biocomposite material to substitute nylon in fishing nets, but it has also been used as part of artificial heart valves.

Figure 1: (a) N. vectensis. (b) Protein sequence. (c,d) N. vectensis images before (c) and after (d) stimulus. Immunohistochemical analyses of N. vectensis with aneroin-specific antibody (e) and purified normal rabbit IgG as a negative control (f). From Yang, Y., Choi, Y., Jung, D., Park, B., Hwang, W., Kim, H. and Cha, H., 2013. Production of a novel silk-like protein from sea anemone and fabrication of wet-spun and electrospun marine-derived silk fibers. NPG Asia Materials, 5(6), pp.e50-e50.

Figure 2: Wet spinning of aneroin fibers: (a) 30KDa aneroin fibers in solution. (b) SEM of wet-spun aneroin-30K fiber surfaces and a breakpoint. (c) Aneroin-30K fiber after spinning. (d) Stress-strain curves of aneroin-30K and aneroin-60K fibers.

Characterization

Optimization of inducer concentration and temperature post-induction

Aim - To determine the concentration of inducer and temperature after induction required for optimal protein expression.

Results - Cell cultures were grown ON at 37°C. Then, the next day, the cultures were diluted to an OD600 of 0.1 and induced with 0.1, 0.3, 0.5 and 1mM IPTG and grew ON at 20, 25, 30 and 37°C. We can clearly see that around 60kDa, there is a darker band in the induced lanes compared to the uninduced lane, showing that the protein is expressed upon induction with IPTG. Further, among the induced lanes, protein expression is maximum when the cultures were incubated at 37°C after induction.

Further, a western blot was done on the above SDS-gel to confirm that the proteins we see are indeed the minispidroin proteins. Since the proteins were expressed with a 6x His-tag, we used mouse anti-hexa his primary antibodies and goat anti-mouse HRP-conjugated secondary antibodies for the western blot.

Conclusion - So, it is clear that induction with 0.3mM IPTG and incubation at 37°C post-induction are the optimal conditions for protein expression.

Protein purification by IMAC

Aim - To purify the protein by IMAC (immobilised metal ion chromatography) using Ni-NTA resin.

Results - Mini columns were loaded with Ni-NTA resin and the soluble fraction of the lysate was added to the columns. Then, the columns were washed twice and eluted to obtain the purified protein. An SDS-gel was run with the different purification fractions and a western blot was done on the gel. We would expect to see a band at 30kDa since that is the expected molecular weight of aneroin. However, in the eluted fraction, we can clearly see a band at 60kDa.

Conclusion - We believe it is probable that the aneroin proteins formed dimers and that is why we see the band of the purified protein at 60kDa, which is twice the expected molecular weight of aneroin (30kDa).

Protein yields

Aim - To do a BCA assay after dialysis of the protein to quantify the final yield.

Results - A 250ml culture of aneroin was induced with 0.3mM IPTG and allowed to grow ON at 37°C to express the proteins. Then, the cells were cultivated and proteins were purified by IMAC using the Ni-NTA resin.

Conclusion - The yield of aneroin was found to be 14 mg/L of culture.

Heat and acid purification

Aim - To purify the aneroin proteins using heat/acid precipitation as an alternative to IMAC using Ni-NTA resin.

Results - It was found by the authors of the aneroin literature that aneroin proteins precipitate at 42°C. Hence, they devised a heat and acid based purification strategy by subjecting the soluble fraction of the cell lysate to 42°C. Then, the lysate was centrifuged to obtain all the aneroin proteins in the pellet. An SDS-gel was run on the supernatants and pellets obtained after subjecting the lysate to 42°C for various durations - 15, 30, 45, 60, 75 and 90mins. A sample that wasn’t subject to heat (0min) was used as controls.

Conclusion - This method did not work since we could not isolate the pure aneroin protein by heat treatment.