Tanaka T, Kimura S, Ota Y
Protein Engineering Research Institute, Toray Industries, Inc., Kanagawa, Japan.
Gene. 1988 Apr 29;64(2):257-64. doi: 10.1016/0378-1119(88)90340-x.
We have developed a phospholipase A2(PLA2)-producing system using Saccharomyces cerevisiae. A 456-bp synthetic DNA fragment was constructed encoding bovine pancreatic phospholipase A2 (proPLA2; zymogen) along with the signal sequence of dog pancreatic PLA2. Yeast-preferred codons were chosen and unique restriction enzyme sites were incorporated. 22 oligodeoxynucleotides that varied in size from 33 to 48 nucleotides were chemically synthesized and assembled into the DNA fragment, which was then placed under the control of the yeast acid phosphatase repressible promoter. The resulting plasmid, transformed into S. cerevisiae, directed the synthesis of about 2.8 micrograms/ml of PLA2, most of which was secreted into the culture fluid. The secreted PLA2 comprised 18 to 26% of active enzyme, the remainder being proenzyme. Both had the expected N-terminal amino acid sequences, indicating that the yeast accurately released the signal peptide and the activation peptide (N-terminal heptapeptide of proPLA2). The specific activity of PLA2 thus produced is the same as that of the authentic bovine enzyme.
我们利用酿酒酵母开发了一种产生磷脂酶A2（PLA2）的系统。构建了一个456碱基对的合成DNA片段，其编码牛胰磷脂酶A2（proPLA2；酶原）以及犬胰PLA2的信号序列。选用了酵母偏好的密码子并引入了独特的限制性酶切位点。化学合成了22个长度从33到48个核苷酸不等的寡脱氧核苷酸，并将它们组装成DNA片段，然后将该片段置于酵母酸性磷酸酶可阻遏启动子的控制之下。将所得质粒转化到酿酒酵母中，指导合成了约2.8微克/毫升的PLA2，其中大部分分泌到培养液中。分泌的PLA2占活性酶的18%至26%，其余为酶原。两者都具有预期的N端氨基酸序列，表明酵母能准确地释放信号肽和激活肽（proPLA2的N端七肽）。由此产生的PLA2的比活性与天然牛酶相同。
