Engineering a Small Molecule-Dependent Intein into a Knottin Cyclotide

Abstract

Peptides are attractive as therapeutics due to their diverse biological function and high potency and target specificity. However, they tend to have short half-lives in vivo due to their susceptibility to degradation by peptidases. Recently, researchers have developed a general strategy to enhance protein stability that involves the use of inherently stable peptides as scaffolds for developing therapeutic proteins. In addition to stability, reliable control of function is desirable for therapeutic proteins for preserving efficiency and minimizing negative side effects; however, this is hard to achieve. Effective posttranslational control of protein expression has been attained for certain proteins using inteins, which are self-splicing protein segments that abolish protein function upon insertion and facilitate the return of function upon excision. Here we aim to combine these efforts and expand the applicability of inteins and knottin scaffolds for therapeutic use by engineering a 4-hydroxytamoxifen (4- HT)-dependent intein into kalata B1 (kB1), a knottin-family cyclotide that possesses exceptional stability due to its cysteine knot structure and has previously been used as a scaffold for engineering new peptide ligands. First, we constructed and expressed kB1 and used high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ ionization (MALDI) mass-spectrometry to differentiate between mature and reduced versions of the peptide. Then, we constructed a knottinN-intein-knottinC fusion construct and analyzed its expression efficiency. Once suitable expression levels are achieved, we plan to induce intein splicing and analyze the mature kB1 product for cysteine knot formation. Successful incorporation of small-molecule dependent inteins into knottin peptides will expand the potential of inteins and knottin peptides for applications in metabolic engineering, drug discovery and delivery, biosensing, molecular computation, and many other areas in biotechnology.