The production of 5-HTP requires the enzyme Tryptophan Hydroxylase 1 (TPH1), which facilitates the conversion of L-tryptophan to 5-HTP. However, TPH1 enzymes tend to aggregate, becoming insoluble and unable to be expressed in E. coli. To address this, we have improved the TPH1 enzyme by converting it into monomeric-human Tryptophan Hydroxylase 1 (m-hTPH1), making it soluble and expressible in E. coli. The activity of m-hTPH1 is supported by its cofactor Tetrahydrobiopterin (BH4), which is regenerated by two coenzymes: hPCBD1 and hQDPR. Therefore, we have decided to express the enzymes m-hTPH1, hPCBD1, and hQDPR in E. coli.
To further enhance 5-HTP production, we fused these three enzymes with different Zinc Finger proteins: Zif268-rigid linker-hTPH1, PBSII-rigid linker-hPCBD1, and ZFa-hQDPR, enabling them to bind to a DNA scaffold via their respective motifs and cluster together. Finally, we verified the expression of these fusion proteins in E. coli strain BL21 to ensure successful protein production.
▲ Figure 1: Induction of protein expression with IPTG and confirmation of fusion protein size by SDS-PAGE.
After preparing the probiotics, we will analyze 5-HTP production using an ELISA kit. We will provide the results once the kit has arrived.