Lee Ji-Hye, Lee Ji-Eun, Kang Kyung-Jung, Jang Young-Joo
Department of Nanobiomedical Science & BK21 PLUS Global Research Center for Regenerative Medicine, Dankook University, 29 Anseo-Dong, Cheonan, 330-714, South Korea.
Department of Nanobiomedical Science & BK21 PLUS Global Research Center for Regenerative Medicine, Dankook University, 29 Anseo-Dong, Cheonan, 330-714, South Korea.
Protein Expr Purif. 2017 Jul;135:37-44. doi: 10.1016/j.pep.2017.05.001. Epub 2017 May 6.
Fibroblast growth factor (FGF) is a multifunctional growth factor that induces cell proliferation, survival, migration, and differentiation in various cell types and tissues. With these biological functions, FGF-2 has been evaluated for clinical use in the regeneration of damaged tissues. The expression of hFGF-2 in Escherichia coli and a purification system using the immobilized metal affinity chromatography (IMAC) is well established to generate a continuous supply of FGF-2. Although hexa-histidine tag (H) is commonly used for IMAC purification, hexa-histidine-asparagine tag (HN) is also efficient for purification as it is easily exposed on the surface of the protein. In this study, four different tagging constructs of hFGF-2 based on tag positions and types (H-FGF2, FGF2-H, HN-FGF2, and FGF2-HN) were designed and expressed under the inducible T7 expression system in E. coli. The experimental conditions of expression and purification of each recombinant protein were optimized. The effective dosages of the recombinant proteins were determined based on the increase of cell proliferation in human gingival fibroblast. ED50s of H-FGF2, FGF2-H, HN-FGF2, and FGF2-HN were determined (4.42 ng/ml, 3.55 ng/ml, 3.54 ng/ml, and 4.14 ng/ml, respectively) and found to be comparable to commercial FGF-2 (3.67 ng/ml). All the recombinant hFGF-2s inhibit the osteogenic induction and mineralization in human periodontal ligament-derived cells. Our data suggested that biological activities of the recombinant hFGF-2 are irrelevant to types and positions of tags, but may have an influence on the expression efficiency and solubility.
成纤维细胞生长因子（FGF）是一种多功能生长因子，可诱导多种细胞类型和组织中的细胞增殖、存活、迁移和分化。凭借这些生物学功能，FGF-2已被评估用于受损组织再生的临床应用。在大肠杆菌中表达hFGF-2并使用固定化金属亲和色谱（IMAC）的纯化系统已得到充分确立，以持续供应FGF-2。尽管六组氨酸标签（H）通常用于IMAC纯化，但六组氨酸-天冬酰胺标签（HN）也因其易于暴露在蛋白质表面而对纯化有效。在本研究中，基于标签位置和类型设计了四种不同的hFGF-2标签构建体（H-FGF2、FGF2-H、HN-FGF2和FGF2-HN），并在大肠杆菌的诱导型T7表达系统下进行表达。对每种重组蛋白的表达和纯化实验条件进行了优化。根据人牙龈成纤维细胞中细胞增殖的增加确定了重组蛋白的有效剂量。测定了H-FGF2、FGF2-H、HN-FGF2和FGF2-HN的半数有效剂量（分别为4.42 ng/ml、3.55 ng/ml、3.54 ng/ml和4.14 ng/ml），发现与市售FGF-2（3.67 ng/ml）相当。所有重组hFGF-2均抑制人牙周膜来源细胞中的成骨诱导和矿化。我们的数据表明，重组hFGF-2的生物学活性与标签的类型和位置无关，但可能对表达效率和溶解性有影响。
