Part:BBa_K2417007
Winsulin - Single Chain Insulin Analogue
What are single chain insulins?
The newest frontier in synthetic insulin analogues is single-chain insulin analogues. They consist of the same A and B chains but instead of a C-peptide they contain a short linker peptide, that is usually flexible and may raise the pI of the protein closer to physiological pH. Due to the small size and high flexibility of the linker peptide, there is no requirement for cleavage and the insulin can still fold into its normal physiological structure, without obstruction.
Currently studied single chain insulin analogues have markedly increased thermodynamic stability, do not require cleavage of the C-peptide and so significantly reduce the complexity of purification procedures.
There are many inventive uses arising from this new structure including Lactococcus secretion of an acid-stable insulin for potential oral administration. All of these new functions are dependent on the linker peptide used.
Our design
The A and B chains (Figure 1) are modelled off human insulin chains, so as to avoid immunogenicity, except for a Glycine residue substitution (A21G) to increase stability of the peptide by increasing the overall pI.
The linker peptide we have designed is QRGGGSGGGQKR, which was designed to avoid encroaching on other published single chain insulin designs whilst still maintaining flexibility, a short length, and basic residues that increase the pH of the protein.
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.
Figure 1: Our Winsulin construct as depicted in SnapGene, consisting of no cleavage sites unlike a full proinsulin.
The design of this linker peptide Increases the pI of protein from 5.6 to 7.78. It has been proven that current long-lasting insulin analogues on the market have a pI of close to neutral pH. We do not have research into how this will last in the human body, as there have been no human trials of single-chain insulin analogues (and especially not our Winsulin!) but we believe it will result in a longer-lasting peptide, and will be a more thermostable alternative to standard human proinsulin.
Characterisation We have shown that our single chain insulin analogue is similar enough to human insulin in structure that it will bind to anti-insulin antibodies, by performing an ELISA whereby the capture antibodies were specific to insulin. We tested multiple types of Winsulin (with various secretion tags), but all were found to bind and induce a reaction (Figure 1).
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.
Furthermore, we tested this part for bioactivity by testing a construct of it (YncM Winsulin - part BBa_K2417005) on an insulin-sensitive cell line, HepG2 cells (human liver cells) (Figure 3). We showed that upon treatment with YncM Winsulin, these cells showed higher rates of glycogen synthesis than basal levels, and showed glucose oxidation activity above basal level in HepG2 cells. These results are excellent preliminary data suggesting bioactivity, which may be confirmed with further testing.
Figure 3: Figure 3: HepG12 cells were incubated with cell lysates expressing Proinsulin/Winsulin for 4 hours in media containing D-[U-14C] glucose, which was then used to analyse glucose oxidation and glycogen synthesis through measurement of 14-CO2 and 14C glycogen production respectively. Treated cells were compared against untreated controls, which were considered to produce basal levels of glycogen synthesis and glucose oxidation. Cells treated with YncM-Winsulin showed higher rates of glycogen synthesis than those at basal levels, and an increase in glucose oxidation was observed. Significance calculated using an unpaired one-tailed T test, n=3, where * is p<0.05. Error bars represent SEM.
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.
(5) Vajo, Z., Fawcett, J. & Duckworth, W. 2001, "Recombinant DNA Technology in the Treatment ofDiabetes: Insulin Analogs", Endocrine Reviews, vol. 22, no. 5, pp. 706-717
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]
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