Part:BBa_K2417002
Ecotin-Winsulin
Winsulin (part BBa_K2417007) with the addition of an N-terminal Ecotin tag (part BBa_K2417010). The use of the Ecotin tag induces the bacteria to express the protein of interest into the periplasm, which has been shown to be a favourable cellular compartment for the folding of disulphide bonds, such as those found in Winsulin. Furthermore, Ecotin has been identified as a model protein for the export of proinsulin into the periplasm of E. coli.
This part is also tagged on the N-terminus with a 6x His tag (part BBa_K2417008), simplifying purification procedures. Between the His tag and Ecotin tag a GGS(x4) linker can be found (part BBa_K2417004), used to expose the His tag for more efficient purification. Finally, the addition of a TEV protease site allows cleavage of the His tag from the purified protein post-purification. Upstream of the protein coding region is an extended ribosome binding site (part BBa_K2417009) chosen for its efficiency. The entire structure of this part has been visualised using SnapGene (Figure 1) for clarification.
Figure 1: Pictorial representation of Ecotin-Winsulin sequence in SnapGene, to indicate the location of each basic part.
Characterisation
We have produced an SDS-PAGE gel of cell lysates after cleavage of His tag and N-terminal secretion tags, and the C-peptide of proinsulins, meaning all insulins/Winsulins were approximately 11kDa (Figure 2).
Figure 2: SDS-PAGE gel (4-20% acrylamide) run at 200V for 1 hour, depicting Cytoplasmic Proinsulin, Cytoplasmic Winsulin, Ecotin Proinsulin, Ecotin Winsulin, YncM Proinsulin (part not submitted) and YncM Winsulin. All proteins were sourced from complete cell lysates obtained by first lysing via addition of lysozyme and freezing, followed by bead beating. Test proteins were treated with proteases to remove N-terminal tags (TEV protease for Winsulin constructs, and trypsin for proinsulin constructs). Negative control proteins are lysed cells that have not been treated with proteases. Insulin proteins without N-terminal tags should be present in ~11kDa band.
For the design of Winsulin, we would like to thank Prof. Peter Arvan from the University of Michigan for allowing us to use his work on single-chain insulins (reference #3) as a primary scaffold for our design, and for providing the foundational information that first inspired us to pursue such a project.
For more detailed information on each of the basic parts comprising this composite parts, please see their individual pages.
References:
(1) Malik, A., Jenzsch, M., Lübbert, A., Rudolph, R. & Söhling, B. 2007, "Periplasmic production of native human proinsulin as a fusion to E. coli ecotin", Protein Expression and Purification, vol. 55, no. 1, pp. 100-111.
(2) Winter, J., Neubauer, P., Glockshuber, R. & Rudolph, R. 2000, "Increased production of human proinsulin in the periplasmic space of Escherichia coli by fusion to DsbA", Journal of Biotechnology, vol. 84, no. 2, pp. 175-185.
(3) Rajpal, G., Liu, M., Zhang, Y. & Arvan, P. 2009, "Single-Chain Insulins as Receptor Agonists", Molecular Endocrinology, vol. 23, no. 5, pp. 679-688.
(4) Hua, Q., Nakagawa, S.H., Jia, W., Huang, K., Phillips, N.B., Hu, S. & Weiss, M.A. 2008, "Design of an active ultrastable single-chain insulin analog: Synthesis, structure, and therapeutic implications", Journal of Biological Chemistry, vol. 283, no. 21, pp. 14703-14716.
(5) Mao, R., Wu, D., Hu, S., Zhou, K., Wang, M. & Wang, Y. 2017, "Secretory expression and surface display of a new and biologically active single-chain insulin (SCI-59) analog by lactic acid bacteria", Applied Microbiology and Biotechnology, vol. 101, no. 8, pp. 3259-3271.
(6) Kohn, W.D., Micanovic, R., Myers, S.L., Vick, A.M., Kahl, S.D., Zhang, L., Strifler, B.A., Li, S., Shang, J., Beals, J.M., Mayer, J.P. & DiMarchi, R.D. 2007, "pI-shifted insulin analogs with extended in vivo time action and favorable receptor selectivity", Peptides, vol. 28, no. 4, pp. 935-948.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |