Difference between revisions of "Part:BBa K4247010"

Latest revision as of 15:37, 12 October 2022

Minispidroin_NT-2rep-CT_SnoopTag_N-6His

This part codes for the full minispidroin protein, a highly soluble spider silk protein, with a 6x His-tag in the N-terminus and the SnoopTag in the C-terminus. This is a composite part consisting of the following basic parts: BBa_K4247005, BBa_K4247001, BBa_K4247002 and BBa_K4247008

This part is one of a collection of compatible minispidroin parts: BBa_K4247000 (Minispidroin_NT), BBa_K4247001 (Minispidroin_2rep), BBa_K4247002 (Minispidroin_CT), BBa_K4247004 (Minispidroin_NT-2rep-CT), BBa_K4247005 (Minispidroin_NT_N-6His), BBa_K4247007 (Minispidroin_NT-2rep-CT_N-6His), BBa_K4247010 (Minispidroin_NT-2rep-CT-SnoopTag_N-6His), BBa_K4247011 (Minispidroin_NT-4rep-CT), BBa_K4247012 (Minispidroin_NT-4rep-CT_N-6His), BBa_K4247013 (Minispidroin_NT-4rep-CT-SnoopTag_N-6His).

Usage and Biology

Dragline silk produced by spiders is one of the strongest natural materials to exist and it is mainly made up of structural proteins called spidroins. These spidroins consist of non-repetitive N-terminal and C-terminal domains and a repetitive central part consisting of tandem repeats of a certain amino acid sequence. These sequences are rich in alanine and glycine to form the crystalline and amorphous parts of the fibre respectively.

There are many research articles whose authors could successfully produce recombinant spider silk proteins and spin them into fibres by mimicking the conditions of the spider’s silk gland where the fibers are formed naturally. But a major drawback in many of these recombinant spidroins was their low solubility. It has been found that the N-terminus of the spidroin is highly soluble at neutral pH which contributes to the solubility of the protein.

In the spider's silk gland, before spinning, the spidroins remain in a highly concentrated and soluble state. Then, this highly concentrated spidroin solution called spinning dope is subject to a gradual drop in pH from 7.6 to 5.7 along the gland which triggers the formation of the fiber. This drop in pH triggers the N-terminus to be more stable and form large network-like structures whereas the C-terminus becomes more unstable to drive spontaneous fibre formation by forming the beta-sheet fibrils which form the core of the fiber. The N-terminal domain restricts the formation of silk fibers to a precise point in the silk duct, preventing silk proteins stored in the silk gland from agglutinating.

This clearly shows us that the solubility and pH sensitivity have a huge effect on the N- and C-terminus of the spidroin which thus affects the formation of fibers. It has been found that the N-terminus of MaSp1 (Major ampullate spidroin 1) from Euprosthenops australis, shows extremely high solubility and pH sensitivity whereas the C-terminus has low solubility and is inert to pH changes and vice versa for the MiSp (Minor ampullate spidroin) of Araneus ventricosus.

Andersson et al., 2017 show how minispidroin can be spun into long fibers

BBa_K4247010 contains the coding sequence for the full minispidroin protein with 2 repeats of the central repetitive domain, a 6x His-tag in the N-terminus and a SnoopTag in the C-terminus. The SnoopTag enables spontaneous isopeptide bond formation with any other protein containing SnoopCatcher (BBa_K4247009) thus enabling easy and modular polyprotein construction.

Characterization

Addition of SnoopTag to Minispidroin_NT-2rep-CT_N-6His

Aim - To attach the sequence coding for SnoopTag to the protein minispidroin_NT-4rep-CT_N-6His via PCR and produce the proteins.

Results -

Conclusion - We can clearly see the protein with the SnoopTag (34.6kDa) since the band is slightly higher than that of the protein without the SnoopTag (30kDa)

Protein purification by IMAC

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

Results - Ni-NTA resin was added to the soluble fraction of the lysate and allowed to incubate for 1h under shaking conditions. Then, it was centrifuged to collect the resin with the proteins and the supernatant was collected as flowthrough. Then, the resin was washed twice and eluted twice. We would expect to see a band at around 34.6kDa since that is the molecular weight of the protein.

Conclusion - Hence, it is clear that the protein with the SnoopTag is eluted in high amounts, good purity and without much losses in the flowthrough or washes.

Simulating the assembly mechanism silk fiber networks

We simulated the formation of a spider silk fiber network during spinning and stretching of the silk fiber using dissipative particle dynamics. This was done on a different number of the characteristic hydrophobic alanine-repeat motif. The data from these simulations was then analysed by identifying clusters of the hydrophobic parts of the repeats and generalising them into nodes (red dots), and links between nodes was then indicated in bridges (black lines), where their thickness indicates the number of connections.

The simulation results indicate that spider silk proteins with 3 and 4 repeats seem to form the most stable fiber networks with large average beta sheet crystals and many connecting bridges, although our simulations were limited because we did not get a chance to simulate proteins with a larger number of repeats than 6.

Fig: Graph depicting the development of average beta-sheet crystals size and number of bridges for networks of spider silk proteins with different number of repeats. The average beta-sheet crystals size is measure in the number of hydrophibic beads that are included in beta-sheets, where each bead represent 3 hydrophobic amino acid residues.

Additionally we simulated spinning under the conditions of failed spinning attempts to try and troubleshoot and fix our mistakes for future attempts. We observed that the failed spinning case that we examined with a protein concentration of 15% had a lowered size of its average beta sheet crystals as well as a low connectivity. Through an additional simulation, with raised shear rate, which represents a higher shear force in spinning, accomplished by a narrower needle, it was observed that a raised shear rate could counteract the lowered average size of beta sheet crystals, but not the network connectivity.

Fig: Graph depicting the development of average beta-sheet crystals size and number of bridges for networks of spider silk proteins with different number of repeats, at different concentrations of spinning, and with different shear rates, which indicate how much shear force is applied to the proteins during spinning (lack of specific shear rate indicates a shear rate of 0.1). The average beta-sheet crystals size is measure in the number of hydrophibic beads that are included in beta-sheets, where each bead represent 3 hydrophobic amino acid residues.