使用带有 L-甲状腺素配体的温敏聚合物对人血清白蛋白进行亲和沉淀。
Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand.
机构信息
State Key Laboratory of Bioreactor Engineering, Department of Bioengineering, East China University of Science and Technology, 130 Meilong Rd,, Shanghai 200237, China.
出版信息
BMC Biotechnol. 2013 Dec 17;13:109. doi: 10.1186/1472-6750-13-109.
BACKGROUND
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. 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.
RESULTS
A 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.
CONCLUSION
A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum.
背景
亲和沉淀已被报道为一种用于下游加工早期蛋白质纯化的潜在技术。该技术可以使用可逆的可溶-不可溶聚合物与亲和配体结合，从大量稀释的溶液材料（如发酵液或血浆）中纯化蛋白质。在这项研究中，使用 N-羟甲基丙烯酰胺、N-异丙基丙烯酰胺和丙烯酸丁酯作为单体合成了一种温敏聚合物。用粘度法测量的聚合物分子量为 3.06×104 Da，最低临界溶液温度（LCST）为 28.0°C。LCST 以上聚合物的回收率超过 95.0%。人血清白蛋白（HSA）是人血清系统中最丰富的蛋白质，在人体中具有重要功能。在制药中需要高纯度的 HSA。安全高效的纯化是 HSA 生产过程中的关键步骤。
结果
合成了一种温敏聚合物，并将 L-甲状腺素固定在聚合物上作为亲和配体，用于 HSA 的亲和沉淀。亲和聚合物的 LCST 为 31.0°C，经过三次循环回收后，其原始量的回收率为 99.6%。最佳吸附条件为 0.02 M Tris-HCl 缓冲液（pH 7.0），亲和沉淀过程中 HSA 的吸附容量为 14.9 mg/g 聚合物。圆二色光谱和 ForteBio Octet 系统用于分析吸附和解吸过程中亲和聚合物与 HSA 之间的相互作用。用 1.0 mol/L NaSCN 洗脱，总 HSA 的回收率为 93.6%。当亲和聚合物应用于人血清中 HSA 的纯化时，根据 SDS-PAGE 可以将 HSA 纯化至单带纯度。
结论
合成了一种温敏聚合物，并将 L-甲状腺素连接到聚合物上。亲和沉淀用于从人血清中纯化 HSA。
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