Part:BBa_K2417003
Cytoplasmic-Winsulin
Winsulin protein (part BBa_K2417007) with the addition of an N-terminal His tag (part BBa_K2415008) and an extended ribosome binding site chosen for its efficiency (part BBa_K2417009). Has a GGS(x4) linker peptide (part BBa_K2417004) between the His tag and protein to allow flexibility and better His tag binding to affinity chromatography column. Also contains a TEV protease site (part BBa_K2417012) between His tag and protein, in order to allow cleavage of tag and release of functional protein. The structure of this part was visualised using SnapGene (Figure 1).
Figure 1: Pictorial representation of Cytoplasmic-Winsulin sequence in SnapGene, to indicate the location of each basic part.
Characterisation
We characterised this part by showing its expression in the cytoplasmic fraction of E. coli cells through detection in an ELISA assay (results of which are shown in Figure 2) where we detected 0.49ng/mL Cytoplasmic Winsulin.
Figure 2: ELISA assay confirms correct folding and structure of proinsulin and Winsulin constructs. ELISA assay was performed on cell lysates for all constructs (additionally, we tested the surrounding media for YncM Winsulin), and showed the presence of Proinsulin or Winsulin in cells expressing Cytoplasmic Proinsulin, Cytoplasmic Winsulin, Ecotin Proinsulin and YncM Winsulin. The assay also proved their ability to bind anti-insulin antibodies, thus suggesting proper folding and structure. 5µL of cell lysates were tested at multiple dilutions, shown here are the 1:1 dilutions.
We also 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 3).
Figure 3: 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 #4) 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 part, please see each individual part page.
References: (1) 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.
(2) 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.
(3) 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.
(4) Rajpal, G., Liu, M., Zhang, Y. & Arvan, P. 2009, "Single-Chain Insulins as Receptor Agonists", Molecular Endocrinology, vol. 23, no. 5, pp. 679-688.
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 |