TY - JOUR
T1 - Towards Enhanced Tunability of Aqueous Biphasic Systems
T2 - Furthering the Grasp of Fluorinated Ionic Liquids in the Purification of Proteins
AU - Carvalho, Sara F.
AU - Custódio, Margarida H.
AU - Pereiro, Ana B.
AU - Araújo, João M. M.
N1 - info:eu-repo/grantAgreement/FCT/3599-PPCDT/PTDC%2FEQU-EQU%2F2223%2F2021/PT#
info:eu-repo/grantAgreement/FCT//SFRH%2FBD%2F139677%2F2018/PT#
info:eu-repo/grantAgreement/FCT/CEEC IND 3ed/2020.00835.CEECIND%2FCP1586%2FCT0014/PT#
info:eu-repo/grantAgreement/FCT/CEEC IND4ed/2021.01432.CEECIND%2FCP1657%2FCT0044/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/LA%2FP%2F0008%2F2020/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F50006%2F2020/PT#
info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F50006%2F2020/PT#
Funding Information:
This research was funded by FCT/MCTES (Portugal) through the project PTDC/EQU-EQU/2223/2021 (DOI 10.54499/PTDC/EQU-EQU/2223/2021).
Funding Information:
This research was supported by FCT/MCTES (Portugal), through grant SFRH/BD/139677/2018 (S.F.C.), and the Individual Call to Scientific Employment Stimulus 2020.00835.CEECIND (J.M.M.A.) and 2021.01432.CEECIND (A.B.P.). This work also received support from FCT/MCTES (LA/P/0008/2020 DOI https://doi.org/10.54499/LA/P/0008/2020 , UIDP/50006/2020 DOI https://doi.org/10.54499/UIDP/50006/2020 and UIDB/50006/2020 DOI https://doi.org/10.54499/UIDB/50006/2020 ), through national funds.
Publisher Copyright:
© 2024 by the authors.
PY - 2024/5/25
Y1 - 2024/5/25
N2 - This work unfolds functionalized ABSs composed of FILs ([C2C1Im][C4F9SO3] and [(Formula presented.)][C4F9SO3]), mere fluoro-containing ILs ([C2C1Im][CF3SO3] and [C4C1Im][CF3SO3]), known globular protein stabilizers (sucrose and [(Formula presented.)][C4F9SO3]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K3PO4). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs’ biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [C2C1Im][C4F9SO3] + 2 %wt K3PO4 and 30 %wt [C2C1Im][C4F9SO3] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity.
AB - This work unfolds functionalized ABSs composed of FILs ([C2C1Im][C4F9SO3] and [(Formula presented.)][C4F9SO3]), mere fluoro-containing ILs ([C2C1Im][CF3SO3] and [C4C1Im][CF3SO3]), known globular protein stabilizers (sucrose and [(Formula presented.)][C4F9SO3]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K3PO4). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs’ biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [C2C1Im][C4F9SO3] + 2 %wt K3PO4 and 30 %wt [C2C1Im][C4F9SO3] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity.
KW - aqueous biphasic systems
KW - biocompatible fluorinated ionic liquids
KW - BSA
KW - lysozyme
KW - selective partition
UR - http://www.scopus.com/inward/record.url?scp=85195832763&partnerID=8YFLogxK
U2 - 10.3390/ijms25115766
DO - 10.3390/ijms25115766
M3 - Article
C2 - 38891953
AN - SCOPUS:85195832763
SN - 1661-6596
VL - 25
JO - International Journal of Molecular Sciences
JF - International Journal of Molecular Sciences
IS - 11
M1 - 5766
ER -