Protein purification by chemo-selective precipitation using thermoresponsive polymers

Area of Science:

• Bioconjugation Chemistry
• Protein Purification
• Polymer Science

Background:

• Purification of proteins with specific functional groups, like free sulfhydryl groups, remains a challenge.
• Developing methods that protect protein functionality during purification is crucial.

Purpose of the Study:

• To synthesize a recoverable and thermoresponsive polymer-protein bioconjugate.
• To utilize this bioconjugate for the chemo-selective purification of proteins containing free sulfhydryl groups.
• To demonstrate the preservation of protein functionality after purification.

Main Methods:

• Modification of target protein's cysteine residue with a disulfide-containing initiator.
• Grafting of poly(N-isopropylacrylamide) (PNIPAM) from the protein, creating a thermoresponsive bioconjugate.
• Thermoprecipitation for separation and purification of the target protein.
• Enzyme activity assays to confirm protein functionality post-purification.

Main Results:

• Successful synthesis of a thermoresponsive polymer-protein bioconjugate.
• Demonstrated thermoprecipitation and separation of target proteins (e.g., bovine serum albumin) with a recycling yield of 76.4%.
• Verified that reversible polymer modification protected papain enzyme activity under extreme conditions.

Conclusions:

• The developed polymer-protein bioconjugate offers an effective strategy for protein purification.
• This chemo-selective enrichment method shows potential for isolating and purifying proteins while maintaining their biological activity.
• The approach is suitable for proteins with free sulfhydryl groups and can be applied under various environmental conditions.