Novel Method for the Isolation of Proteins and Small Target Molecules from Biological and Aqueous Media by Salt-Assisted Phase Transformation of Their PEGylated Recognition Counterparts

Javad Mokhtari; Maryam Nourisefat; Bita Zamiri; Leila Fotouhi; Amir-Hassan Zarnani; Ali Akbar Moosavi-Movahedi; Khashayar Karimian

doi:10.1021/acsomega.0c06149

Novel Method for the Isolation of Proteins and Small Target Molecules from Biological and Aqueous Media by Salt-Assisted Phase Transformation of Their PEGylated Recognition Counterparts

ACS Omega. 2021 Mar 10;6(11):7585-7597. doi: 10.1021/acsomega.0c06149. eCollection 2021 Mar 23.

Authors

Javad Mokhtari^{1

2}, Maryam Nourisefat³, Bita Zamiri³, Leila Fotouhi³, Amir-Hassan Zarnani^{4

5}, Ali Akbar Moosavi-Movahedi³, Khashayar Karimian^{1

3}

Affiliations

¹ Arasto Pharmaceutical Chemicals Inc., Yousefabad, Jahanarar Avenue, 23rd St. No. 8, Tehran 1438933741, Iran.
² Departments of Chemistry, Science and Research Branch, Azad University, Tehran 1477893855, Iran.
³ Institute of Biochemistry and Biophysics, University of Tehran, Tehran 1417466191 Iran.
⁴ Department of Immunology, School of Public Health, Tehran University of Medical Sciences, Tehran 1417466191, Iran.
⁵ Reproductive Immunology Research Center, Avicenna Research Institute, ACECR, Tehran 1936773493, Iran.

Abstract

An efficient and simple method for the application of PEGylated affinity ligands in precipitative isolation of protein target molecules (TMs) from a biological fluid such as blood serum or small target molecules from an aqueous medium is presented for the first time. This approach is based on the high binding specificity of PEGylated recognition molecules (PEG-RMs) to their TMs and the unique physicochemical properties of PEG that result in their salt-assisted phase transformation. Addition of PEG-RM to blood serum results in the formation of an RM-specific macromolecular complex (PEG-RM + TM → PEG-RM.TM) that undergoes facile salt-assisted phase transformation to a separable semisolid with ammonium sulfate. PEG-RM.TM is then dissociated into its components by pH reduction or an increase of ionic strength (PEG-RM.TM → PEG-RM + TM). PEG-RM is salted out to afford pure TM in solution. The same phenomenon is observed when RM or TM are small molecules. The general applicability of the method was validated by PEGylation of two proteins (protein A, sheep antihuman IgG) and a small molecule (salicylic acid) used as model RMs for the isolation of Igs, IgG, and serum albumin from blood serum. The isolated protein TMs were shown to be pure and aggregate-free by gel electrophoresis and dynamic light scattering (DLS). IgG isolated by this method was further characterized by peptide mass fingerprinting. PEGylated protein A was used to demonstrate the recyclability and scale-up potential of PEG-RM. IgG isolated by this method from blood serum of a hepatitis C-vaccinated individual was tested for its binding to sheep antihuman IgG by UV spectroscopy, and its bioactivity was ascertained by comparison of its enzyme-linked immunosorbent assay (ELISA) result to that of a blood sample from the same individual. Reciprocity of RM and TM was ascertained using PEGylated salicylic acid to obtain pure serum albumin, and PEGylated serum albumin was utilized for near-exclusive isolation of one drug from an aqueous equimolar mixture of three drugs (salicylic acid, 91%; capecitabine, 6%; and deferiprone, 3%). Advantages of this approach, including target specificity and general applicability and celerity, over other affinity methods for the isolation of proteins are discussed at a molecular level.