Part:BBa_K1731001:Experience

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Applications of BBa_K1731001

Insulin lispro is a human insulin analog that dissociates more rapidly than human regular insulin after subcutaneous injection, resulting in higher insulin levels at an earlier point in time and a shorter duration of action[1, see References un Design section]. We designed a sequence of constitutive expression for insulin lispro. Our aim is to express lispro as proinsulin containing peptide C and with a TAT sequence in its N terminus. This construct is expected to be expressed in Rossetagami and SHuffle E. coli strains, where correct eukaryotic protein expression and folding is enhanced. We expect to have a fully formed insulin with disulphide bonds.

This part could be used to improve insulin production at small and large scale; the promoter could also be modified in order to express it in other organisms or in order to make it signal-dependent.

Within basic research interests, the part could become a model for protein folding molecular dynamics due to insulin's way of folding by itself in particular oxidative environments.

Some biotechnology applications include the way we are willing to use it as a source for an insulin-dependent diabetic patient. In the future we would replace the promoter of this construct for an OmpC promoter so that its expression can be modulated by EnvZ activation.

User Reviews

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This part was easily assembled because it was synthesized as a single fragment. It's product may be hard to work with due to protein's accumulation and precipitation when it is not properly folded or when it is expressed in extremely large quantities.

It has been described in the literature that it is hard for insulin to form its disulphide bonds, a reaction highly dependent in the environment oxidative capacities. Even by synthesizing the primary sequence and treating the peptide after its purification it is hard to form the correct disulphide bonds in recombinant insulin production for medication purposes.

We could express the primary sequence of the protein and we detected it by western blot, but we are waiting to have much more product to analyze it by proteolysis assays as well as mass spectrometry in order to characterize whether the disulphide bonds formed or not, and if they did, whether they formed corectl or not.

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