Kuroda S, Miyazaki T, Otaka S, Fujisawa Y
Biotechnology Research Laboratories, Takeda Chemical Industries, Ltd., Osaka, Japan.
Appl Microbiol Biotechnol. 1993 Nov;40(2-3):333-40. doi: 10.1007/BF00170389.
We constructed a plasmid that directs the synthesis and secretion of hepatitis B virus (HBV) surface antigen (HBsAg) particles by Saccharomyces cerevisiae. This plasmid contains a proteinase-resistant HBsAg M (M-P31c) gene fused at its 5'-terminus with a chicken-lysozyme signal peptide (C-SIG) gene, which is placed under the yeast GLD (glyceraldehyde-3-phosphate dehydrogenase gene) promoter. The products encoded by the "C-SIG+M-P31c" (LM-P31c) gene were synthesized and assembled themselves into HBsAg particles in yeast cells, and the particles were released into the medium along with poly-HSA (polymerized human serum albumin) binding activity. The HBsAg particles purified from the medium were very similar in density (1.19 g cm-3), size (19.2 +/- 0.8 nm in diameter) and shape (sphere) to human-plasma-derived HBsAg particles. When several sec (temperature-sensitive secretion-defective) mutants were used as host cells, the release of HBsAg particles into the medium was blocked at 37 degrees C but not at 25 degrees C, indicating that the HBsAg particles are exported through the normal yeast secretion pathway. To our knowledge, this is the first report that yeast cells are capable of secreting particles into the medium.
我们构建了一种质粒，该质粒可指导酿酒酵母合成并分泌乙型肝炎病毒（HBV）表面抗原（HBsAg）颗粒。此质粒包含一个蛋白酶抗性的HBsAg M（M-P31c）基因，其5'端与鸡溶菌酶信号肽（C-SIG）基因融合，该信号肽基因置于酵母甘油醛-3-磷酸脱氢酶基因（GLD）启动子的控制之下。由“C-SIG+M-P31c”（LM-P31c）基因编码的产物在酵母细胞中合成并组装成HBsAg颗粒，这些颗粒与多聚人血清白蛋白（poly-HSA）结合活性一起释放到培养基中。从培养基中纯化得到的HBsAg颗粒在密度（1.19 g/cm³）、大小（直径19.2±0.8 nm）和形状（球形）方面与源自人血浆的HBsAg颗粒非常相似。当使用几种sec（温度敏感型分泌缺陷）突变体作为宿主细胞时，HBsAg颗粒在37℃时释放到培养基中的过程受阻，但在25℃时不受阻，这表明HBsAg颗粒是通过正常的酵母分泌途径输出的。据我们所知，这是关于酵母细胞能够将颗粒分泌到培养基中的首次报道。
