A novel protein purification scheme based on salt inducible self-assembling peptides

Table 1 Application of the icSAT scheme for the purification of different proteins and peptides

Type	Target protein	Intein	0.7 M Na₂SO₄		3 M NaCl		0.7 M (NH₄)₂SO₄		Specific activity of purified proteins and peptides	Specific activity of commercial or literature-sourced proteins and peptides
Type	Target protein	Intein	Yield^a (mg/L)	Purity^b (%)	Yield^a (mg/L)	Purity^b (%)	Yield^a (mg/L)	Purity^b (%)	Specific activity of purified proteins and peptides
Therapeutic protein	hGH	Mtu ΔI-CM	87 ± 6	97 ± 1	81 ± 12	94 ± 7	127 ± 16	88 ± 7	0.13–0.19 nM^c	0.15 nM^d
Peptide	LCB3	Mtu ΔI-CM	12 ± 3	72 ± 1	10 ± 6	71 ± 16	4 ± 3	33 ± 8	0.31–0.43 nM^c	< 1 nM [32]
Multivalent scaffold protein	SpyCatcherΔN-ELP-SpyCatcherΔN	Mtu ΔI-CM	40 ± 9	96 ± 1	45 ± 8	98 ± 1	40 ± 6	85 ± 4	~ 99%^e	~ 80%^f [34]
Industrial enzyme	xylanase	Mtu ΔI-CM	32 ± 4	79 ± 1	24 ± 4	67 ± 3	18 ± 1	59 ± 4	12.37–26.19 units/mg	≥ 2.50 units/mg^g
Industrial enzyme	xylanase	Mxe GyrA	84 ± 6	87 ± 6	35 ± 13	49 ± 15	37 ± 6	54 ± 7	13.31–28.60 units/mg	≥ 2.50 units/mg^g

^aYield of target proteins or peptides after intein-mediated cleavage per liter of culture. ^bPurity is calculated as the mass ratio of target proteins or peptides to total proteins in the supernatant after intein-mediated cleavage, estimated using densitometry analysis software ImageJ. ^cThe dissociation constants of hGH for binding to the hGH receptor and of LCB3 for binding to the SARS-CoV-2 spike receptor were measured using the respective commercial receptors. Prior to the binding assays, the three icSAT-purified hGH proteins and LCB3 peptides were subject to ion-exchange chromatography to achieve further purification. ^dThe dissociation constant of commercial hGH for binding to the hGH receptor in this study. ^eThe covalent reconstitution between SpyCatcherΔN-ELP-SpyCatcherΔN and LCB3-SpyTag was measured using a 3-fold excess of LCB3-SpyTag, following the protocol recommended by Paul J Wichgers Schreur [35]. ^fThe covalent reconstitution was that between SpyCatcher and SpyTag-MBP. ^gThe activity of xylanase available commercially from Sigma-Aldrich

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