Date of Award

2025

Type

Thesis

Major

Chemistry

Degree Type

Bachelor of Science in Chemistry

Department

Chemistry

First Advisor

Dr. Jonathan Meyers

Second Advisor

Dr. Stephanie DaSilva

Third Advisor

Dr. Ansley Felix

Abstract

Preptin, a 34-amino-acid peptide derived from proinsulin-like growth factor II, has emerged as a promising therapeutic candidate for both Type 2 diabetes mellitus and osteoporosis. Its dual functionality—stimulating insulin secretion without inducing hypoglycemia and promoting osteoblast proliferation—positions it as a unique alternative to traditional sulfonylureas and osteogenic agents. In this study, we developed a purification protocol for green fluorescent-preptin fusion proteins. These fusion proteins will be used to investigate the functional significance of residues Pro17, Val18, Gly19, Trp27, Lys28, and Gln29 in rat preptin, which shares high sequence similarity with the human form. Site-directed mutagenesis was performed using NEB Q5 QuickChange methodology, followed by recombinant expression in Escherichia coli. The GFP-preptin fusion protein was purified using nickel affinity chromatography, followed by gel electrophoresis to confirm molecular weight and purity. The use of GFP facilitated real-time tracking of purification efficiency, with SDS-PAGE revealing a distinct 27 kDa band corresponding to the target fusion protein. Lyophilization enabled long-term storage without loss of integrity. These findings establish a robust platform for the expression and purification of preptin analogs, supporting future efforts to characterize their biochemical activity and therapeutic potential in glycemic control and bone regeneration.

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