Yee C J, DeFrances M C, Bell A, Bowen W, Petersen B, Michalopoulos G K, Zarnegar R
Department of Pathology, School of Medicine, University of Pittsburgh, Pennsylvania 15261.
Biochemistry. 1993 Aug 10;32(31):7922-31. doi: 10.1021/bi00082a013.
A cDNA containing the entire coding sequence of human hepatocyte growth factor (HGF) [also known as scatter factor (SF)] was inserted into the genome of Autographa california nuclear polyhedrosis virus (baculovirus) adjacent to the polyhedrin promoter by homologous recombination. Insect cells (Spodoptera frugiperda) infected with the recombinant virus secrete relatively high levels (3-8 mg/L) of biologically active HGF into the culture medium. The recombinant HGF induces pronounced morphological changes and scattering of primary cultures of rat, mouse, and human hepatocytes within 24 h after plating and stimulates DNA synthesis in these cells with the same magnitude as native HGF derived from human placenta or rabbit serum. The human recombinant HGF produced by the insect cells is N-glycosylated, binds to heparin like native HGF, and is recognized by polyclonal antiserums raised against human or rabbit HGF as assessed by immunoblot, ELISA, and immunoneutralization experiments. Metabolic radiolabeling with L-[35S]methionine (pulse-chase experiments) as well as Western blot analysis indicates that the recombinant HGF is synthesized and secreted by the infected insect cells as the unprocessed single-chain form (pro-HGF) when the cells are cultured in serum-free medium. However, when the infected insect cells are cultured in insect culture medium (Grace's medium) containing fetal bovine serum, the secreted HGF is present mainly in the mature heterodimeric form. Addition of serum to the baculovirus-expressed single-chain [125I]HGF in a cell-free system results in conversion to the heterodimeric two-chain form, and the activation is prevented by the serine protease inhibitor PMSF. Incubation of 125I-labeled pro-HGF with rat liver or spleen extracts resulted in conversion of pro-HGF to the heterodimeric two-chain form. A truncated form of HGF containing the N-terminal portion of HGF (kringles 1-3) was also produced in the same expression system. This deleted HGF, by itself, did not have any detectable biological activity; however, it abrogated the stimulatory effects of full-length HGF on hepatocytes. This is the first successful production of bioactive recombinant HGF in large quantities, which will allow purification on the milligram scale of pro-HGF and will permit future studies to elucidate pathways involved in HGF activation by its target tissues.
通过同源重组，将包含人肝细胞生长因子（HGF）[也称为分散因子（SF）]完整编码序列的cDNA插入到苜蓿银纹夜蛾核型多角体病毒（杆状病毒）基因组中多角体蛋白启动子附近。用重组病毒感染昆虫细胞（草地贪夜蛾）后，可将相对高水平（3 - 8毫克/升）的生物活性HGF分泌到培养基中。重组HGF在接种后24小时内可诱导大鼠、小鼠和人原代肝细胞培养物发生明显的形态变化并使其分散，且能刺激这些细胞中的DNA合成，其刺激程度与源自人胎盘或兔血清的天然HGF相同。昆虫细胞产生的人重组HGF进行了N - 糖基化修饰，能像天然HGF一样与肝素结合，并且通过免疫印迹、酶联免疫吸附测定和免疫中和实验评估，可被针对人或兔HGF产生的多克隆抗血清识别。用L - [³⁵S]甲硫氨酸进行代谢放射性标记（脉冲追踪实验）以及蛋白质免疫印迹分析表明，当在无血清培养基中培养时，重组HGF以未加工的单链形式（前体HGF）由被感染的昆虫细胞合成并分泌。然而，当在含有胎牛血清的昆虫培养基（格雷斯培养基）中培养被感染的昆虫细胞时，分泌的HGF主要以成熟的异二聚体形式存在。在无细胞体系中，向杆状病毒表达的单链[¹²⁵I]HGF中添加血清会导致其转化为异二聚体双链形式，且丝氨酸蛋白酶抑制剂苯甲基磺酰氟（PMSF）可阻止这种激活。用¹²⁵I标记的前体HGF与大鼠肝脏或脾脏提取物一起孵育会导致前体HGF转化为异二聚体双链形式。在同一表达系统中还产生了一种截短形式的HGF，其包含HGF的N端部分（kringles 1 - 3）。这种缺失的HGF本身没有任何可检测到的生物活性；然而，它消除了全长HGF对肝细胞的刺激作用。这是首次成功大量生产生物活性重组HGF，这将使得能够以毫克规模纯化前体HGF，并将有助于未来研究阐明其靶组织激活HGF所涉及的途径。
