Methods Of Intracellular Conversion Of Single-Chain Proteins Into Their Di-Chain Form

摘要

The present specification discloses expression constructs comprising single-chain proteins comprising a di-chain loop region comprising an exogenous protease cleavage site and a protease that can cleave the exogenous protease cleavage site located within the di-chain loop, cell compositions comprising such expression construct, and intracellular methods of converting the single-chain protein into its di-chain form.

主权项

1. An intracellular method of converting a single-chain Clostridial toxin into its di-chain form, the method comprising the steps of:
a) growing a cell comprising a dual expression construct at 37° C. for about 3.5 hours, the dual expression construct comprising;
i) an open reading frame encoding a single-chain Clostridial toxin, the single-chain Clostridial toxin comprising an enzymatic domain, a translocation domain, a binding domain, and a di-chain loop region comprising an exogenous protease cleavage site; andii) an open reading frame encoding a protease; wherein the protease can cleave the exogenous protease cleavage site located within the di-chain loop; b) growing the cell at 22° C. for about 16 to about 18 hours,
wherein growth at step (b) induces expression of the single-chain Clostridial toxin and the protease from the dual expression construct; andwherein the produced protease cleaves the single-chain Clostridial toxin at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain Clostridial toxin into its di-chain form.