Target tag | PHA synthase-intein-target | PhaP-intein-target [11] | PhaP-intein-target [12] | PhaP-intein-target [13] | PhaR-intein-target [14] |
---|---|---|---|---|---|
Association with beads | Covalently attached in vivo in E. coli | Non-covalently attached in vivo in E. coli | Non-covalently attached in vivo in R. eutropha | In vitro non-covalent association | In vitro non-covalent association |
Intein/activate condition | Ssp DnaB intein/low pH | ∆I-CM engineered from Mtu recA intein/low pH | Mxe GyrA intein/thiols | Ssp DnaB intein/low pH | Ssp DnaB intein/low pH |
Efforts on extra protein/polymer production | No concerns | Extra triple or dual PhaP protein production | Extra PhaP production | Extra separate chemical synthesis of PHB polymer | Extra separate chemical synthesis of PHB polymer |
Tailoring efforts on washing/elution conditions to prevent PhaP/PhaF leaching | No concerns | Tailoring efforts needed for an intermediate salt content in washing/elution | No discussion but necessary to prevent PhaP leaching while only allow target elution | No discussion but necessary to prevent PhaP leaching while only allow target elution | No discussion but necessary to prevent PhaR leaching while only allow target elution |
Limitation on purifying a potentially bead associated target | No concerns (except for PHA synthase tag itself) | With limitations, e.g. β-lactamase unsuitable | No discussion but with limitations in order to maintain non-covalent PhaP: bead binding | With limitations, e.g. PHA synthase unsuitable | No discussion but with limitations in order to maintain non-covalent PhaR:bead binding |
Potentially multiple short cleavage cycles | Exemplified with PHA synthase-intein-GFP beads | Unexplored | Unexplored | Unexplored | Unexplored |
Purification for therapeutic proteins containing S–S bond | Explored for TNFα, IFNα2b, and G-CSF | unexplored | Unsuitable due to use of thiols | unexplored | unexplored |