Part:BBa_K2417005
YncM-Winsulin
Consists of Winsulin (part BBa_K2417007) with an N-terminal translocation tag (YncM – part BBa_K2417011) to induce secretion of the protein into the surrounding media. Secretion of a protein removes the need for complex extraction techniques, and so simplifies the production process.
An N-terminal His tag (part BBa_K2417008) was also added in order to facilitate purification using an affinity chromatography column. Finally, the extended ribosome binding site (part BBa_K2417009) upstream of the protein coding region was chosen for its high efficiency.
Finally, the addition of a TEV protease site (part BBa_K2417012) between the His tag and protein allows cleavage of the histidine chain after purification. A pictorial representation of the part was generated on SnapGene (see Figure 1).
Figure 1: Pictorial representation of YncM-Winsulin sequence in SnapGene, to indicate the location of each basic part.
Characterisation
The part was characterised through performing an ELISA assay to demonstrate that YncM-Winsulin was found in the surrounding media of the cells, but not in the whole cell lysate (Figure 2).
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. The inclusion of a whole-cell lysate control for YncM Winsulin proves the YncM secretion tag was effectively causing Bacillus secretion of Winsulin into the surrounding media, as it was not found in the cell lysate fraction.
Furthermore, we carried out an assay on insulin-sensitive cells to identify bioactivity. We showed that in the presence of YncM-Winsulin cells had increased glycogen synthase activity and increased glucose oxidation, indicating a similar bioactivity to unmodified insulin (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 a slight 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.
Finally, we 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 more information on each of the basic parts comprising this composite part, please see each individual basic part page.
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 #2) as a primary scaffold for our design, and for providing the foundational information that first inspired us to pursue such a project.
References: (1) J. Olmos-Soto, R. Contreras-Flores, Genetic system constructed to overproduce and secrete proinsulin in Bacillus subtilis, Appl. Micro- biol. Biotechnol. 62 (2003) 369 – 373.
(2) 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]
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