Engineering, expression, and renaturation of a collagen-targeted human bFGF fusion protein
Engineering, expression, and renaturation of a collagen-targeted human bFGF fusion protein
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DOI：
10.3109/08977190109029115
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发表时间：
2001-01-01
期刊：
影响因子：
1.8
通讯作者：
Becerra, J
中科院分区：
文献类型：
--
作者：
Andrades, JA;Wu, LT;Becerra, J
碱性成纤维细胞生长因子(碱性成纤维细胞生长因子)在体外是一种有效的毛细血管内皮细胞有丝分裂原，在体内刺激血管生成，并可能参与组织修复。碱性成纤维细胞生长因子广泛存在于脑、肾和软骨等组织中。本研究报道了一种具有生物活性的人碱性成纤维细胞生长因子融合蛋白的表达、纯化和复性。通过构建原核表达载体，构建了由三部分组成的融合蛋白：(1)纯化标签；(2)蛋白酶敏感连接区/胶原结合区；(3)编码人碱性成纤维细胞生长因子活性片段的c DNA序列。在不同的实验条件下，表达的hbFGF-F1和hbFGF-F2(含一个胶原结合区)以包涵体形式存在，用6M盐酸胍溶解，用谷胱甘肽氧化还原系统复性，并进行长时间的透析。通过Ni-NTA金属螯合柱结合融合蛋白的组氨酸标签，实现了重组蛋白的纯化。用[H-3]-胸腺嘧啶核苷掺入法检测重组生长因子对人静脉内皮细胞增殖的影响，并以商用重组人碱性成纤维细胞生长因子(RhbFGF)为阳性对照，验证重组生长因子的生物学活性。纯化的重组人碱性成纤维细胞生长因子-F1和重组人碱性成纤维细胞生长因子-F2载体显示出与商品化重组碱性成纤维细胞生长因子相当的增殖活性。复性的hbFGF-F2融合蛋白在胶原连接的Sephadex-G15柱上稳定结合。[H-3]-胶原与固定在Ni-NTA柱上的重组人碱性成纤维细胞生长因子-F2蛋白的结合也证明了这种高亲和力。重组人碱性成纤维细胞生长因子-F2融合蛋白以高亲和力结合在胶原涂层表面，但其生物学活性低于溶液中的融合蛋白，提示其具有潜在的潜在构象。综上所述，这些结果表明，通过氧化复性可以从转化细菌中回收具有生物活性的重组人碱性成纤维细胞生长因子融合蛋白，从而为其从微生物中高产生产、纯化和复性提供了手段。此外，我们证明了辅助胶原结合域有效地将重组生长因子靶向I型胶原。此外，还研究了重组人碱性成纤维细胞生长因子F2对链脲佐菌素诱导的糖尿病大鼠创面愈合的临床作用，并通过组织学检查，与重组人碱性成纤维细胞生长因子F1和商品碱性成纤维细胞生长因子的作用进行了比较。重组人碱性成纤维细胞生长因子-F2对创面加热的高度有益作用可能是由于其极强的血管生成和肉芽组织形成活动，导致创面快速再上皮化。局部应用重组人碱性成纤维细胞生长因子-F2和I型胶原是一种加速糖尿病大鼠全层皮肤切除创面愈合的更有效的方法，与单用融合蛋白或相同剂量的商品化hbFGF相比。这些研究推进了产生大量靶向bFGF融合蛋白所需的技术，以及为特定的生物医学应用开发新的策略。
Basic fibroblast growth factor (bFGF) is a potent in vitro mitogen for capillary endothelial cells, stimulates angiogenesis in vivo, and may participate in tissue repair. Basic FGF is found in abundance in tissues such as brain, kidney and cartilage. This study reports the expression, purification, and renaturation of a biologically active human basic fibroblast growth factor fusion protein (libFGF-F1) from Escherichia coli. A prokaryotic expression vector was engineered to produce a tripartite fusion protein consisting of (i) a purification tag, (ii) a protease-sensitive linker/collagen-binding domain, and (iii) cDNA sequence encoding the active fragment of hbFGF. The expressed hbFGF-F1 and hbFGF-F2 (it contains a collagen-binding domain), located in inclusion bodies, were solubilized with 6 M guanidine-HCl and renatured using a glutathione redox system and protracted dialysis under various experimental conditions. The purification of the recombinant proteins was achieved by binding the His-tag of the fusion protein on a Ni-NTA metal chelate column. The biological activity of the recombinant growth factors was demonstrated by their ability to stimulate proliferation of human vein endothelial cells (HVEC), monitored by [H-3]-thymidine incorporation, where commercial recombinant human bFGF (rhbFGF) served as a positive control. Purified rhbFGF-F1 and rhbFGF-F2 constructs exhibited proliferative activity comparable to commercial rbjbFGF. Binding of the renatured hbFGF-F2 fusion protein to collagen was demonstrated by stable binding on a collagen-conjugated Sephadex-G15 column. The high affinity binding was also demonstrated by the binding of [H-3]-collagen to the rhbFGF-F2 protein immobilized on a Ni-NTA column. The rhbFGF-F2 fusion protein bound to collagen coated surfaces with high affinity but exhibited comparatively lower biological activity than the fusion protein in solution, suggesting a potentially latent configuration. Taken together, these results demonstrate that biologically active rhbFGF fusion proteins can be recovered from transformed bacteria by oxidative refolding; thus, providing a means for its high-yield production, purification, and renaturation from microorganisms. Furthermore, we demonstrate that the auxiliary collagen-binding domain effectively targets the recombinant growth factor to type I collagen. The clinical effect of rhbFGF-F2 on wound healing is also studied in streptozotocin-induced diabetic rats and evaluated by histological examination comparing with rhbFGF-F1 and commercial bFGF effects. The highly beneficial effects of rhbFGF-F2 on wound heating is suggested to be due to its extremely potent angiogenesis and granulation tissue formation activities, leading to a rapid reepithelialization of the wound. Topical application of rhbFGF-F2 mixed with type I collagen is a more effective method in accelerating closure of full-thickness excisional skin-wound in diabetic rats when compared with the fusion protein alone or commercial hbFGF at the same doses. These studies advance the technology necessary to generate large quantities of targeted bFGF fusion proteins as well as to develop new strategies for specific biomedical applications.