Part:BBa_K2417001
Cytoplasmic-Proinsulin
Consists of the human proinsulin coding region (part BBa_K2417006), sourced from NCIB, preceded by an efficient extended ribosomal binding site (part BBa_K2417009) and an N-terminal His tag (part BBa_K2417008). The His tag was added to allow ease of purification, using affinity chromatography. A GGS(x4) linker (part BBa_2417004) was created to expose the His tag during affinity chromatography, with a trypsin protease cleavage site (arginine residue) included in order to allow cleavage of His tag post purification. See Figure 1 for a pictorial representation of this part.
Figure 1: Pictorial representation of Cytoplasmic Proinsulin sequence in SnapGene, to indicate the location of each basic part.
Characterisation
We characterised this part by assessing its concentration from cell lysate in an ELISA assay specific to insulin, so confirming its conformation and folding was correct enough to be recognised by an anti-insulin antibody (see 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 Cytoplasmic-Proinsulin, both cell lines showed significantly higher rates of glycogen synthesis than basal levels (Figure 3 and 4), and also showed significantly greater glucose oxidation activity above basal level in HepG2 cells (Figure 3). These results are excellent data confirming bioactivity.
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 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 and glucose oxidation. Cells treated with Cytoplasmic-Proinsulin showed significantly higher rates of glycogen synthesis and glucose oxidation than basal levels. Significance calculated using an unpaired one-tail T test, where * is p<0.05.
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 Cytoplasmic-Proinsulin showed significantly higher rates of glycogen synthesis than those at basal levels. Significance calculated using an unpaired one-tail T test, 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 coding sequence of human proinsulin, which in previous parts was incomplete. Furthermore, the addition of a 6x His tag allows easier purification using an affinity chromatography column, a system generally accessible by most labs.
Parts improved: https://parts.igem.org/wiki/index.php/Part:BBa_M39904 https://parts.igem.org/wiki/index.php/Part:BBa_M1877
Please see basic parts pages for more detailed information on their functions.
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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