Part:BBa_K4944000
mShh with a 6x His-tag (mShh-His)
Mouse (Mus Musculus) Sonic Hedgehog gene (Shh) which codes for mShh protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 695
Illegal NgoMIV site found at 942 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 665
Usage and Biology
Sonic Hedgehog protein is a signaling molecule important in the regulation of embryonic morphogenesis in mammals [1]. The hedgehog family of proteins is notable for their ability to autocatalytically cleave in the presence of cholesterol. While hedgehog (Hh) protein is formed as an inactive precursor, it undergoes an autocatalytic cleavage between Gly-257 and Cys-258 that leads to the creation of two distinct domains: an approximately 19 kDa amino-terminal and a roughly 25 kDa carboxy-terminal domain. The latter domain encompasses essential determinants for the protein's auto-processing capacities, while the former is crucial for its signaling activity. The N-terminal domain becomes cholesterol-modified and the lipid integrates directly with the protein chain [2].
The proteolysis mechanism involves an intramolecular N-to-S acyl transfer on the protein’s backbone, templated by a Hedgehog INTein (Hint) fold (C198-A365). The acyl shift creates a thioester which is intercepted by cholesterol. This causes esterification of the N-terminal’s last residue. The C-terminal Sterol Recognition Region (SRR) (V366-S462) and the Hint domain forms the “Hog” region of the Hedgehog protein, and enables the ligation of cholesterol to the N-terminal domain [3].
In our project, the hedgehog protein's ability to auto-process and add a cholesterol adduct made it the ideal candidate as a component of our cholesterol absorption inhibitor. This gave our fusion protein the ability to bind the sterol binding pocket of transmembrane receptor proteins. This sterol modification holds potential for a variety of design applications. Notably, the Hedgehog protein's ability to auto-process with other molecules, like soy- and 25-hydroxy cholesterol, offers the potential for flexibility and modularity.
References
(1) Merchant JL. Hedgehog signalling in gut development, physiology and cancer. J Physiol 2012;590:421–32. https://doi.org/10.1113/jphysiol.2011.220681.
(2) Rashid MH. Full-length recombinant antibodies from Escherichia coli: production, characterization, effector function (Fc) engineering, and clinical evaluation. MAbs. 2022 Jan-Dec;14(1):2111748. doi: 10.1080/19420862.2022.2111748. PMID: 36018829; PMCID: PMC9423848. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9423848
(3) Porter JA, Ekker SC, Park WJ, Kessler DP von, Young KE, Chen CH, et al. Hedgehog Patterning Activity: Role of a Lipophilic Modification Mediated by the Carboxy-Terminal Autoprocessing Domain. Cell. 1996 Jul 12;86(1):21–34.
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