Incorporation of Aliphatic Proline Residues into Recombinantly Produced Insulin

Abstract

Analogs of proline can be used to expand the chemical space about the residue while maintaining its uniquely restricted conformational space. Here, we demonstrate the incorporation of 4Rmethylproline, 4S-methylproline, and 4-methyleneproline into recombinant insulin expressed in Escherichia coli. These modified proline residues, introduced at position B28, change the biophysical properties of insulin: Incorporation of 4-methyleneproline at B28 accelerates fibril formation, while 4-methylation speeds dissociation from the pharmaceutically formulated hexamer. This work expands the scope of proline analogs amenable to incorporation into recombinant proteins and demonstrates how noncanonical amino acid mutagenesis can be used to engineer the therapeutically relevant properties of protein drugs.

4S-fluorination of ProB29 in insulin lispro slows fibril formation

“…4 A ). Under these conditions, the fibrillation lag time of insulin lispro (10.9 ± 2.2 h) was reduced compared to that of human insulin (16.7 ± 4.1 h) ( 25 ), consistent with the conventional view that fibrillation proceeds from the monomer state ( 11 ). Replacing ProB29 with 4 R -F (10.3 ± 2.4 h) or 44-diF (9.0 ± 1.3 h) did not significantly change the fibrillation lag time.…”

“…Human insulin and lispro variants were prepared as previously described ( 25 ); details are provided in the Supporting information . Incorporation efficiencies for all proline analogs exceeded 90% ( Table S1 ).…”

“…We recently demonstrated that introduction of ncPro residues at position B28 can be used to tune the biophysical properties of human insulin ( 23 , 24 , 25 ). These results prompted us to ask whether similar changes in the biophysical properties of insulin lispro might be achieved.…”

Engineered Proteins and Materials Utilizing Residue-Specific Noncanonical Amino Acid Incorporation

“…While RSI incorporation does permit similar specific structural dynamics to be detected, as demonstrated in the Tirrell group’s work on the incorporation of proline residues into insulin, , its impact on multiple local environments must be considered. As of now, RSI benefits from enabling the generation of highly fluorinated biomolecules that have increased impacts on function and thermostability.…”

Using Chemistry To Recreate the Complexity of the Extracellular Matrix: Guidelines for Supramolecular Hydrogel–Cell Interactions

“…By tuning the elastic sequences and by utilizing RGD as a ligand, materials with specific cellular adhesion signals can be created . Another strategy includes the use of recombinant proteins to fabricate hydrogels, pioneered by the Tirrell lab. , The incorporation of noncanonical amino acids here is very appealing as it allows chemical modification of a specific region of interest in the protein. Overall, the modularity and biocompatibility of protein-based biomaterials makes them good candidates to investigate fundamental cell–matrix processes.…”