Part:BBa_K2417000

Ecotin-Proinsulin

Human proinsulin with the addition of an N-terminal Ecotin tag (part BBa_K2417010). The use of the Ecotin tag allows periplasmic expression, shown to be a favourable cellular compartment for the folding of disulphide bonds, such as those found in proinsulin. Furthermore, Ecotin has been identified as a model protein for the export of proinsulin into the periplasm of E. coli.

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 placement of Arginine sites between the His tag and protein, and within proinsulin, allows a one-step cleavage procedure both removing the His-tag and converting proinsulin into functional insulin.

The protein coding region is preceded by an extended ribosomal binding site (part BBa_K2417009) chosen for its high ribosomal recruitment efficiency. The structure of the part can be visualised in Figure 1.

Figure 1: Pictorial representation of Ecotin Proinsulin sequence using SnapGene program, showing structural features.

Characterisation

We characterised this part by performing an ELISA assay specific to insulin, and through this showing that when tagged at the N-terminus with Ecotin, proinsulin was expressed (through detection in whole cell lysate, as due to time constraints we were unable to test a purified periplasmic fraction) (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.

We also tested this part for bioactivity by testing it on two insulin-sensitive cell lines: HepG2 cells (human liver cells)(Figure 3) and AML-12 (murine liver cells)(Figure 4). We showed that upon treatment with Ecotin-Proinsulin, both cell lines showed significantly higher rates of glycogen synthesis than basal levels (Figure 3 and 4), and showed some glucose oxidation activity above basal level in HepG2 cells (Figure 3). These results are excellent preliminary data suggesting bioactivity, which may be confirmed with further testing.

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 Ecotin-Proinsulin showed significantly 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.

Figure 4: AML-12 cells were incubated with cell lysates expressing Proinsulin/Winsulin constructs for 4 hours in media containing D-[U-14C] glucose, which was then used to measure 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. Cells treated with Ecotin-Proinsulin showed significantly higher rates of glycogen synthesis than those at basal levels. Significance calculated using an unpaired one-tailed T test, n=3, where * is p<0.05 and ** is p<<0.05.

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 5).

Figure 5: 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.

Part Improvement

This part is an improvement of previous insulin parts in the registry, by the addition of the Ecotin signalling peptide and by inclusion of both A and B chains in one coding region. The addition of an efficient ribosome binding site assists future teams in their expression of this protein, and the addition of a His tag aids purification. Furthermore, previous parts only had partial sequences and did not have quantified production.

Parts improved: https://parts.igem.org/wiki/index.php/Part:BBa_M39904 https://parts.igem.org/wiki/index.php/Part:BBa_M1877

For more detailed information on each of the basic parts comprising this composite part, please refer to their individual basic parts 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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]