Part:BBa_K2417006
Human Proinsulin Coding Sequence
Insulin is a key hormone of the human body, normally produced by beta cells of the pancreas, and is required to regulate glucose uptake into tissue from the blood. It is an important pharmacological compound, due to the high prevalence of diabetics dependent on synthetically produced insulin around the world. In the body, it is synthesized as a single polypeptide, preproinsulin (consisting of A, B, C chains and a signal peptide), which is then cleaved in the endoplasmic reticulum to become proinsulin (with signal peptide removed). Here it folds, forming 3 disulphide bonds, and the C-peptide is cleaved and released (Figure 1).
Figure 1: Generation of insulin from proinsulin – in our proinsulin construct the C-peptide (connecting peptide - shown in red) is cleaved using Trypsin, at arginine residues. C-peptide helps with protein folding as it means A (blue) and B (yellow) chain are transported together to the endoplasmic reticulum and can form disulphide bonds.
This gene encodes the coding sequence for human proinsulin, excluding introns, codon optimised for use in E. coli. It also contains arginine residues adjacent to the C-peptide to allow proteolytic cleavage using trypsin (see Figure 2). The sequence for this gene was sourced from NCBI.
Figure 2: Pictorial representation of Proinsulin part as depicted in Snapgene, showing arginine trypsin cleavage sites and A and B chains.
It is an improvement on previous parts submitted (https://parts.igem.org/wiki/index.php/Part:BBa_M1877) and (https://parts.igem.org/wiki/index.php/Part:BBa_M39904) as it contains the A and B chains both in one protein coding sequence, that can easily be cleaved into functional insulin upon treatment with trypsin (cleaves C peptide via adjacent arginine residues). Previously submitted parts either did not contain full sequences or only encoded the A and B chains on separate plasmids, making difficult the synthetic production of a properly folded insulin molecule using bacteria.
References Kemmler, W., Steiner, D.F. & Borg, J. 1973, "Studies on the Conversion of Proinsulin to Insulin", Journal of Biological Chemistry, vol. 248, no. 13, pp. 4544.
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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