Part:BBa_K4247013

Minispidroin_NT-4rep-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. This is a composite part consisting of the following basic parts: BBa_K4247005 (Minispidroin_NT_N-6His), 2 repeats of BBa_K4247001 (Minispidroin_2rep), BBa_K4247002 (Minispidroin_CT) and and BBa_K4247008 (SnoopTag). BBa_K4247012 contains the coding sequence for the full minispidroin protein with 4 repeats of the central repetitive domain and a 6His-tag in the N-terminus of the protein.

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_K247005 (Minispidroin_NT_N-6His), BBa_K247007 (Minispidroin_NT-2rep-CT_N-6His), BBa_K247010 (Minispidroin_NT-2rep-CT-SnoopTag_N-6His), BBa_K247011 (Minispidroin_NT-4rep-CT), BBa_K247012 (Minispidroin_NT-4rep-CT_N-6His), BBa_K247013 (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.

Herein, part BBa_K4247012 is a composite part formed from the following basic parts: BBa_K4247005 (Minispidroin_NT_N-6His), 2 repeats of BBa_K4247001 (Minispidroin_2rep) and BBa_K4247002 (Minispidroin_CT). BBa_K4247012 contains the coding sequence for the full minispidroin protein with 4 repeats of the central repetitive domain, a 6x His-tag in the N-terminus, and the SnoopTag at the C-terminus. The SnoopTag enables spontaneous isopeptide bond formation with any other protein containing SnoopCatcher (part BBa_K247009) thus enabling easy and modular polyprotein construction.

Characterization

Regarding general info on induction, purification, chromatography check BBa_K4247012.

Addition of SnoopTag to minispidroin_NT-4rep-CT_N-6His

Aim - To attach by user PCR the part coding for SnoopTag to the protein minispidroin_NT-4rep-CT_N-6His, clone the protein and produce it in BL21DE3

Results & conclusion-By following the procedure we already used in production of the other minispidroins we managed to express the SnoopTag, which has a MW of 41.8 KDa, compared to the 40.3 KDa of the protein without the tag.