Part:BBa_K2958007
Long Lasting Single Chain Insulin Analog
This single chain proinsulin analog consists of an A Chain and B Chain from Proinsulin (from BBa_K2417006). This insulin has been modified in the A Chain in position 21 (from an Asparagine to an Alanine) to reflect a similar mutation to the patented Long Lasting Insulin, Glargine. Additionally, a GGYLGGGGGGGR linker (BBa_K2958005) has been added in order to get the PI at 6.46, which is close to the PI of the long lasting insulin Glargine (6.7)
Description:
Proinsulin is normally made of an A Chain, B Chain, and C chain. Insulin is in its active form after the C chain is cleaved by endopeptidases. Specifically, prohormone convertases PC1 and PC2. However, with the addition of the GGYLGGGGGGGR linker, the insulin-producing process no longer requires this step. The PI of this single chain insulin is 6.46, which allows for the insulin to exhibit prolonged dissociation when injected into human tissue, and is close in value to the PI of Glargine, a long lasting insulin. The PI of glargine is 6.7. We designed this single chain long lasting insulin with the intent to compare its structure and function to native human insulin in hopes of confirming that the long lasting insulin analog has a slower reaction rate than wild type insulin.
Figure 1. Figure 1. (A) Protein model of native human insulin (active form) produced on Swiss Model software. Orange portion is A chain of insulin, and green portion is B chain of insulin. (B) Protein model of long lasting single chain insulin with GGYLGGGGGGGR linker (BBa_K295005) produced on Swiss Model software. Orange portion is A chain of insulin, green portion is B chain, and red portion is the linker.
Figure 2. Protein model of Native human insulin (orange portion = A chain of native insulin, green portion = B chain of insulin) superimposed on protein model of single chain long lasting insulin (pink portion = A chain of long lasting single chain insulin, blue portion= B chain of long lasting single chain insulin, red portion = GGYLGGGGGGGR linker) produced on Swiss Model Software. Based on these superimposed models alone, it appears that the addition of the linker does not disrupt the structure of the A and B chains. However, the side chains of these insulin molecules require more extensive analysis.
Figure 3. Figure 3. Protein model demonstrating interaction energy (DG) between the single chain native insulin molecule and the ectodomain of the insulin receptor (domain outside of cell membrane). The estimated DG is 1.84606 kcal/mol. In comparison to the estimated DM of our single chain insulin (BBa_K2958006), which was 2.04839 kcal/mol, indicating that the mutations in the long lasting insulin analog decreased the interaction energy by 0.20233 kcal/mol. This model was produced with FoldX software, courtesy of iGEM Team_Moscow 2019.
Figure 4. Protein analysis of the stability of Long Lasting Insulin, conducted by iGEM Team_Moscow with a OPLS AA force field (A.) Analysis of RMSD to check stability of Long Lasting Insulin Analog. The RDSM exhibits a plateau, indicating that this analog is stable. (B) Analysis of the radius of gyration of the long lasting insulin analog to measure compactness. The value of rg is relatively steady, indicating stable protein folding.
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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