Halford Michael M, He Yi-Chao, Stacker Steven A
Tumour Angiogenesis Program, The Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, Australia.
Tumour Angiogenesis Program, The Peter MacCallum Cancer Centre, St Andrews Place, East Melbourne, Victoria, Australia; Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia.
Biotechniques. 2014 Jun 1;56(6):331-3. doi: 10.2144/000114181. eCollection 2014 Jun.
The secreted adhesive glycoprotein vitronectin (VTN) is a multifunctional component of plasma and the extracellular matrix. A high-yielding, inexpensive, low endotoxin source of bioactive recombinant human vitronectin (rhVTN) is highly desirable for in vitro use in diverse cell culture systems ranging from basic research settings to clinical-grade production of human cells. We describe modifications to a previously reported heparin-based affinity chromatography procedure that improve yield and achieve efficient removal of endotoxin from washed and urea-solubilized human VTN inclusion bodies following standard autoinduction of expression in Escherichia coli. This simple procedure makes accessible the low-cost expression and purification of large quantities of bioactive rhVTN using basic equipment and facilitates its use in a spectrum of endotoxin-sensitive applications.
分泌型黏附糖蛋白玻连蛋白（VTN）是血浆和细胞外基质的多功能成分。对于从基础研究到临床级人类细胞生产等多种细胞培养系统的体外应用而言，生物活性重组人玻连蛋白（rhVTN）的高产、廉价、低内毒素来源是非常理想的。我们描述了对先前报道的基于肝素的亲和层析方法的改进，该改进提高了产量，并在大肠杆菌中进行标准自诱导表达后，能从洗涤后的尿素溶解的人VTN包涵体中有效去除内毒素。这个简单的方法使用基本设备就能实现大量生物活性rhVTN的低成本表达和纯化，并便于其在一系列对内毒素敏感的应用中使用。
