Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand

Zhaoyang Ding,Xuejun Cao

doi:10.1186/1472-6750-13-109

Zhaoyang Ding, Xuejun Cao

Open Access

https://doi.org/10.1186/1472-6750-13-109

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Abstract

BackgroundAffinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production.ResultsA thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris–HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE.ConclusionA thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum.

Highlights

Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing
Structure of Human serum albumin (HSA) was shown to decrease from 15.44% (A) to 14.26% (B), 12.44% (C) or 9.08% (D) as in Figure 5 by adding different concentrations of L-thyroxin. These results indicate that the ligand underwent interactions with HSA, and that increasing the amount of L-thyroxin added caused greater changes in the secondary structure of HSA
The results show that the affinity polymers could be repeatedly used for HSA adsorption without any noticeable reduction in recovery

Summary

Introduction

Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. Affinity precipitation was reported as a potential technology for the purification of proteins during early stages of downstream processing [1] This technology could be achieved by using reversibly soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material, such as fermentation broths or plasma. Human Serum Albumin (HSA), the most abundant protein in human plasma, has been one of the most extensively studied proteins over several decades [6,7] It is synthesized in the liver and presents in the blood with a concentration around 40 mg/ml. Dyes [14,15,16,17] and metal ions [18,19,20] are generally used as the affinity ligand to purify HSA

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