Yang Junhao, Kanter Gregory, Voloshin Alexei, Levy Ronald, Swartz James R
Department of Chemical Engineering, Stanford University, Stanford, California 94305, USA.
Biotechnol Prog. 2004 Nov-Dec;20(6):1689-96. doi: 10.1021/bp034350b.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is an important cytokine in the mammalian immune system. It has been expressed in Escherichia coli with the same biological activity as the native protein. Here, we report the synthesis of a murine recombinant GM-CSF in an E. coli cell-free protein synthesis system with a high yield. Since there are two disulfide bonds in the native structure of GM-CSF, an oxidizing redox potential of the reaction mixture was required. By pretreating the cell extract with iodoacetamide (IAM), the reducing activity of the cell extract was inactivated, and upon further application of an oxidized glutathione buffer, most of the synthesized GM-CSF was found in its oxidized form. However, the GM-CSF thus formed showed low activity because of poor folding. With the addition of DsbC, the periplasmic disulfide isomerase from E. coli, a high yield of active GM-CSF was produced in the cell-free reaction. Finally, successful folding of the cell-free synthesized GM-CSF-his6 was confirmed by its cell-proliferation activity after purification with a Ni2+ chelating column.
粒细胞巨噬细胞集落刺激因子（GM-CSF）是哺乳动物免疫系统中的一种重要细胞因子。它已在大肠杆菌中表达，具有与天然蛋白质相同的生物活性。在此，我们报道了在大肠杆菌无细胞蛋白质合成系统中高产合成鼠重组GM-CSF。由于GM-CSF天然结构中有两个二硫键，因此反应混合物需要具有氧化还原电位。通过用碘乙酰胺（IAM）预处理细胞提取物，细胞提取物的还原活性被灭活，进一步应用氧化型谷胱甘肽缓冲液后，发现大部分合成的GM-CSF呈氧化形式。然而，如此形成的GM-CSF由于折叠不良而活性较低。加入来自大肠杆菌的周质二硫键异构酶DsbC后，在无细胞反应中产生了高产率的活性GM-CSF。最后，通过用Ni2+螯合柱纯化后其细胞增殖活性证实了无细胞合成的GM-CSF-his6的成功折叠。
