Improved production of recombinant fibroblast growth factor 7 (FGF7/KGF) from bacteria in high magnesium chloride
Improved production of recombinant fibroblast growth factor 7 (FGF7/KGF) from bacteria in high magnesium chloride
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DOI：
10.1016/j.pep.2003.10.013
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发表时间：
2004-02-01
影响因子：
1.6
通讯作者：
McKeehan, WL
中科院分区：
文献类型：
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作者：
Luo, YD;Cho, HH;McKeehan, WL
由于相对于其他成纤维细胞生长因子 (FGF) 同系物，肝素/硫酸乙酰肝素和上皮细胞上的受体复合物具有特异性，因此人们对 FGF7（也称为角质形成细胞生长因子或 KGF）的临床和商业应用产生了相当大的兴趣，这些应用需要以合理的成本进行大量生产。相对于 FGF1 和 FGF2，从细菌生产重组 FGF7 的产量和回收率较低。 FGF7 在 N 末端与谷胱甘肽-S-转移酶 (GST) 融合，然后通过蛋白水解去除 GST，同时与天然配体肝素结合，将大肠杆菌宿主固有的低产量提高到每 OD600 3.2 毫克/升，仍然仅为 FGF1 的 10%。通过在培养基中包含10-100mM氯化镁(MgCl 2 )，菌株BL21(DE3)pLysS中GST-FGF7融合产物的产量提高至约17mg/L/OD600。从固定在肝素-琼脂糖上的 GST-FGF7 中蛋白水解切除 GST 部分后，这将完全活性 (54)ser-FGF7 的产量提高了约五倍。这种简单的增强提高了在细菌中生产重组 FGF7 用于临床和商业应用的成本效益。 (C) 2003 Elsevier Inc. 保留所有权利。
Because of specificity for both heparin/heparan sulfate and the receptor complex on epithelial cells relative to other fibroblast growth factor (FGF) homologues, there is considerable interest in clinical and commercial applications of FGF7 (also called keratinocyte growth factor or KGF) that require large quantities at reasonable cost. Production of recombinant FGF7 from bacteria suffers from lower yields and recovery relative to FGF1 and FGF2. Fusion of FGF7 at the N-terminus with glutathione-S-transferase (GST) followed by removal of GST by proteolysis while bound to natural ligand heparin improved the intrinsically low yields from Escherichia coli hosts to 3.2 mg per liter per OD600, which was still only 10% of that for FGF1. Yield of the GST-FGF7 fusion product was improved to about 17mg per liter per OD600 in strain BL21(DE3)pLysS by inclusion of 10-100 mM magnesium chloride (MgCl2) in the culture medium. This improved by about five times the yields of fully active (54)ser-FGF7 after proteolytic excision of the GST portion from GST-FGF7 immobilized on heparin-Sepharose. This simple enhancement improves the cost-effectiveness of production of recombinant FGF7 in bacteria for clinical and commercial applications. (C) 2003 Elsevier Inc. All rights reserved.