Preperation of an engineered tryptophan-depleted protein in Pichia pastoris and evaluation of its dietary anti-tumor efficacy.

Targeting amino acid metabolism has emerged as a promising strategy for cancer therapy. To investigate the feasibility of tryptophan restriction as a dietary intervention strategy to suppress tumor growth in vivo, this study developed a novel tryptophan-depleted protein (TDP) through protein engineering combined with the Pichia pastoris expression system, for dietary intervention to suppress tumor growth. Based on the human serum albumin (HSA) scaffold, we designed the TDP by mutating tryptophan at position 238 to phenylalanine (Phe), and successfully expressed it in P. pastoris. Following purification by hollow fiber membrane ultrafiltration and liquid chromatography, High performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis confirmed that the tryptophan content in the purified TDP was only 0.35 μg/g. In a gastric adenocarcinoma xenograft model, the TDP-based tryptophan-restricted diet significantly suppressed tumor growth compared with the control group receiving a complete protein diet (P0.001), resulting in a 64% reduction in tumor weight. This study provides a proof-of-concept for developing engineered dietary proteins as foods for special medical purposes (FSMPs), and establishes a novel strategic framework for dietary intervention in adjuvant cancer therapy.