乳腺生物反应器表达的重组人抗凝血酶Ⅲ的分离纯化

Purification of recombinant human antithrombin Ⅲ expressed in a goat mammary bioreactor

摘要:

抗凝血酶Ⅲ (AT Ⅲ)是人体内最重要的抗凝血物质，利用转基因技术生产重组人抗凝血酶Ⅲ (rhAT Ⅲ)受到了越来越多的关注，如何有效地将rhAT Ⅲ从转基因羊奶中分离纯化出来是实现其产业化的关键所在。本研究建立了基于等电点沉淀和肝素亲和层析的纯化工艺，实现了rhAT Ⅲ的高效、快速纯化。先用等电点沉淀法，快速去除了占总蛋白50%左右的酪蛋白；然后用肝素亲和层析纯化rhAT Ⅲ，并系统考察了pH值和温度对rhAT Ⅲ稳定性的影响，以及pH值和操作条件 (洗脱梯度、流速和上样量) 对rhAT Ⅲ分离效果的影响。在优化的条件下，rhAT Ⅲ纯度达到99%以上，蛋白收率达到90%，活性收率约为50%。所建立的纯化工艺简单、快速，rhAT Ⅲ收率高，为今后工艺放大提供了重要的依据和参考。

Abstract:

Antithrombin III (AT Ⅲ) is the most important anti-clotting substance. Recombinant human antithrombin Ⅲ (rhAT Ⅲ) expressed in transgenic goat milk attracts more and more attention. Develop an effective purification route for rhAT ⅢI is vital to its industrial production. An efficient purification method was developed for the rapid purification of rhAT Ⅲ by isoelectric precipitation and heparin affinity chromatography.?First, casein was effectively removed by isoelectric precipitation. rhAT Ⅲ was further purified by heparin affinity chromatography.?In the process of heparin affinity chromatography, the effects of pH and temperature on the stability of rhAT Ⅲ were studied, and the effects of operating conditions, elution gradient, flow rate and sample loaded, on the purification efficiency were also studied.?Under the optimized conditions, the protein recovery of rhAT Ⅲ was about 90% with purity over 99%, while its activity recovery was about 50%. Such a purification process?is?very simple and effective, and it would provide a valuable reference for the further scaling-up of industrial production.