2024 AIChE Annual Meeting

Engineering a More Efficient De Novo Protein for Quantum Dot Synthesis

Quantum dots (QDs) are semiconductor nanocrystals with optical properties similar to fluorescent dyes but offer longer fluorescent durations and higher photon emission. These properties make QDs suitable for bioimaging and cancer cell-tagging. However, common QDs like cadmium sulfide (CdS) are potentially toxic for in vivo applications. Non-toxic alternatives, such as biomineralized CuInS₂ quantum dots, are being explored. Traditionally, the protein cystathionine γ-lyase (CSE) synthesizes QDs by breaking down cysteine into H₂S, pyruvate, and ammonia, which react with metals to form nanocrystals. Recently, de novo protein Construct K (ConK), designed through computational models, was discovered to catalyze QD synthesis in a similar manner. Although ConK can produce QDs, its reaction time is significantly longer (10+ hours) compared to CSE (30 minutes).

To address this, I investigated mutations in ConK to accelerate the synthesis process. I focused on mutating aromatic amino acids near the active site, specifically histidine residues at positions 43 and 46, to alanine or lysine. Site-directed mutagenesis was performed on ConK plasmids, followed by transformations into DH5α E. coli cells for plasmid amplification. Sequencing confirmed successful mutations in H43K and H46A. These plasmids are now being transformed into BL21 E. coli cells for protein overexpression.

The next phase involves purifying the mutated ConK proteins and testing their ability to synthesize non-toxic QDs. The ultimate goal is to enhance the reaction rate and scale up QD production for industrial and medical applications, making cancer diagnostics and treatments more accessible.