Difference between revisions of "Part:BBa K4339009"

Latest revision as of 13:21, 12 October 2022

MaSp2 6Rep

A major ampullate spidroin 2 (MaSp2) based protein consisting of six repetitive regions from the black widow spider Latrodectus hesperus. Each repetitive region begins with a glycine rich region and ends with between seven and nine alanines.

Usage and Biology

Sequence and Features

The sequence entirely originates from the black widow spider Latrodectus hesperus using repetitive domains from [1] and [2].

All MaSp parts (K4339002 - K4339009) can be categorised as either MaSp1 or MaSp2 proteins, which form the major components of spider dragline silk. Both protein types comprise of repeated poly-alanine and glycine rich regions. Hydrophobic interactions between poly-alanine regions and hydrophilic interactions between glycine-rich regions confer the strength and elasticity of dragline silk respectively [3]. The non-repetitive N- and C-terminal domains of these proteins are suggested to contribute towards protein solubility and self-assembly in the abdominal glands of spiders, where proteins are exposed to a pH gradient as they are drawn through an abdominal duct [4]. Following the protocol for spontaneous fibre assembly from MaSps outlined in [5], which occurs in the absence of a pH gradient, the N-terminal domains, which only contribute to fibre assembly via interactions with such a gradient, would be non-functional, so was removed from all MaSp constructs.

Results reported in [5] show the number of repeated poly-alanine and glycine rich regions controls fibre length, ranging from millimetre to metre scale. Results further show that removing the C-terminal domain causes formation of an amorphous aggregate of short fibres. Thus, MaSp constructs were designed containing various numbers of repeated regions, with and without C-terminal domains, with an aim to generate fibres of a range of structures, to explore the resultant differences in their mechanical properties.

In silico studies predict that MaSp1 and MaSp2 proteins have distinctly different mechanical behaviour, with the overall mechanical properties of a silk fibre being partially dependent on the ratio of the two protein types [6]. MaSp1 is predicted to contribute more greatly to elasticity of a silk fibre, whilst MaSp2 is predicted to contribute more significantly to its Young's modulus (a measure of a material's resistance to deformation). Therefore, to explore the range of possible mechanical properties of recombinant spider silk, both MaSp1 and MaSp2 parts were designed, with an aim to express and purify both protein types, before mixing them in a variety of ratios and triggering their spontaneous self-assembly into fibres.

All MaSp parts were designed to be integrated into devices, fused with a His tag (6 x Histidine) and solubility tag TrxA (BBa_K3619001) with a thrombin cleavage site between them. TrxA is a small, highly soluble protein which, when fused with a protein of interest, can act as a molecular chaperon, guiding protein folding and preventing inclusion body formation [7], as well as increasing the solubility of the fusion partner. As per the methodology set out in [5], TrxA-fused MaSp proteins were designed to be soluble in the cytosol of E. coli when expressed, so they could be extracted in the soluble portion cell lysate. MaSps would be purified from the lysate using a Nickel-NTA column (which binds his-tagged proteins). Thrombin would then be added to the elution from the column to cleave off the TrxA tag, causing MaSp proteins to precipitate out of solution and spontaneously self-assemble into fibres.

MaSp2 6Rep was successfully inserted into a holding plasmid and sequence verified, but with a large region of the coding sequence missing. A His-tagged device containing this part was built in a medium-copy plasmid vector under the control of the inducible T7 promoter and sequence verified (with only 4 point mutations and 1 base insertion). A TrxA+His-tagged device containing this part was built similarly and transformed into Rosetta DE3 E. coli, but expression could not be confirmed via Western blotting.

References

[1] - Ayoub, N et al. Blueprint for a High-Performance Biomaterial: Full-Length Spider Dragline Silk Genes. PLoS ONE. 2007;2(6). doi: https://doi.org/10.1371/journal.pone.0000514

[2] - You, Z. et al. Extraordinary Mechanical Properties of Composite Silk Through Hereditable Transgenic Silkworm Expressing Recombinant Major Ampullate Spidroin. Scientific Reports. 2018;8(1): 1–14. doi: https://doi.org/10.1038/s41598-018-34150-y

[3] - Xia X et al. Native-sized recombinant spider silk protein produced in metabolically engineered Escherichia coli results in a strong fiber. Proc Natl Acad Sci USA. 2010;107(32): 14059-63. doi: https://doi.org/10.1073/pnas.1003366107

[4] - Gaines W et al. Recombinant Dragline Silk-Like Proteins-Expression and Purification. AATCC Rev. 2011;11(2): 75-79. Available from: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3729042/ [Accessed 12/10/22]

[5] - Stark M et al. Macroscopic fibers self-assembled from recombinant miniature spider silk proteins. 2007;8(5): 1695–1701. doi: https://doi.org/10.1021/bm070049y.

[6] - Brooks A et al. Distinct contributions of model MaSp1 and MaSp2 like peptides to the mechanical properties of synthetic major ampullate silk fibers as revealed in silico. Nanotechnol Sci Appl. 2008;1: 9-16. doi: https://doi.org/10.2147/nsa.s3961

[7] - LaVallie E et al. Thioredoxin as a fusion partner for production of soluble recombinant proteins in Escherichia coli. Methods in Enzymology. 2000;326: 322-340. doi: https://doi.org/10.1016/S0076-6879(00)26063-1