The invention relates to an in vitro cell-free expression system incorporating a novel inorganic polyphosphate-based energy regeneration system. In certain embodiments, the invention includes a cell-free expression system where the cellular energy source, ATP, is regenerated from inorganic polyphosphate using a dual enzyme system. In this embodiment, this dual enzyme system may include thermostable Adenosyl Kinase, and/or Polyphosphate Kinase enzymes.

Compositions and methods of modulating cellular function and treatment of disease in mammals comprising locally administering a regulated SNARE inhibitor and a translocating agent to the mammal. Regulated SNARE inhibitors include clostridial neurotoxins, tetanus neurotoxin and their free light chain portions and IgA protease. Translocating agents include acids, encapsulating vectors, and transduction domains.

The present disclosure discloses a method for purifying tetanus toxin or variants thereof. The method for purifying tetanus toxin or variants thereof uses metal chelate affinity chromatography, and the tetanus toxin does not contain an affinity tag. The present disclosure uses biocompatible agarose-based nickel affinity chromatography and anion exchange chromatography as separation subjects, which can quickly and effectively remove impure proteins and enrich target proteins; it improves the separation and purification efficiency of proteins and greatly reduces the loss of proteins.