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Table 5 Comparision of the PhaC based protein purification method with previous methods based on PhaP/PhaR tag

From: Purification of target proteins from intracellular inclusions mediated by intein cleavable polyhydroxyalkanoate synthase fusions

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