Methods of intracellular conversion of single-chain proteins into their di-chain form
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.
1. An intracellular method of converting a single-chain protein into its di-chain form, the method comprising the steps of:
(a) growing a cell comprising a dual expression construct at a first temperature for a certain period of time in order to achieve maximal cell density, the dual expression construct comprising:
(i) an open reading frame encoding the single-chain protein comprising a di-chain loop region comprising an exogenous protease cleavage site; and
(ii) 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 a second temperature for a certain period of time in order to achieve maximal induction of protein expression from the open reading frame encoding the single-chain protein,
wherein growth at step (b) induces expression of the single-chain protein and the protease from the dual expression construct; and
wherein the produced protease cleaves the single-chain protein at the exogenous protease cleavage site located within the di-chain loop region, thereby converting the single-chain protein into its di-chain form.
2. The method according to claim 1 , wherein the exogenous protease cleavage site is an enterokinase protease cleavage site, a human rhinovirus 3C protease cleavage site, a human enterovirus 3C protease cleavage site, a Tobacco Vein Mottling Virus (TVMV) protease cleavage site, a subtilisin protease cleavage site, or a Caspase 3 protease cleavage site.