Severe chronic and sometimes incurable diseases are frequently accompanied by a pain syndrome. Purotoxin-1, isolated from the poison of the Central Asian Geolycosa sp. spider and selectively inhibiting the purinergic P2X3 receptor (which is considered as a target for the control of the pain states), is one potentially highly effective drug with an analgesic effect. To produce the recombinant purotoxin-1, we created four genetically engineered constructions with different carrier proteins for the expression in E. coli: pTRX-PT1, pCBD-PT1, pGyrA-PT1, pDnaB-PT1. The construction with mini-intein DnaB from the Synechocystis sp. was the most efficient. Using the E. coli C3030/pDnaB-PT1 producer strain, the laboratory method, based on which a pilot technology of recombinant purotoxin-1 production was developed as a result of optimization and scaling, was created. Six grams of recombinant PT1 preparation with the confirmed pharmacological purity was developed for preclinical trials.