EXPRESSION OF EUKARYOTIC DNA REPLICATION PROTEIN COMPLEXS AND THEIR SIMPLE PURIFICATION METHOD

真核DNA复制蛋白复合物的表达及其简单纯化方法

• 批准号: 07558101
• 负责人: SUGINO Akio
• 金额: $ 13.63万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (A)
• 财政年份: 1995
• 资助国家: 日本
• 起止时间: 1995 至 1996
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07558101/
• 关键词: DNA Replication protein complex; Expression vector; Baculovirus expression vector; Insect cells; Budding yeast; Cdc7; Dbf4 protein kinase complex; RF-C complex; DNA polymerase complex; YACベクター; Cdc7-Dbf4複合体; RFC複合体

In last year, we have succeeded in expressing yeast Saccharomyces cerevisae Cdc7/Dbf4 protein kinase complex which is required for the initiation of yeast chromosomal DNA replication using Vaculovirus vector in Sf9 insect cells. In this year, we tried to develop a new and simple purification method of Cdc7/Dbf4 protein complex from Sf9 cells infected with the recombinant virus containing CDC7 and DBF4 genes using Mcm2 protein as a kinase substrate. Using MonoQ,hydroxylapatite, and Heparin-Sepharose columns, we were able to purify the protein complex. Using the purified Cdc7/Dbf4 protein kinase complex, we found that the kinase phosphorylates not only S.cerevisiae Mcm2 protein, but also Mcm3, Mcm4, Mcm6, and Dbf4 protein. However, Mcm proteins expressed in and purified from E.coli were not good substrates for the kinase, suggesting that the kinase prefers partially phosphorylated proteins.Using yeast expression vector containing Gal 1 and Gal10 promotors, we were able to express yeast R … More F-C complex consisting of five different subunits (Rfc1p (Cdc44p), Rfc2p, Rfc3p, Rfc4p and Rfc5p) in yeast. In the presence of galactose, yeast cells expressed at least 50 time more RF-C activity than in the absence of galactose. Since PCNA stimulates the ATPase activity of RF-C complex, RF-C complex should tightly interact with PCNA.Based un this, we made PCNA-conjugated Sepharose column. Using this column, we were able to obtain more than mg of highly purified RF-C complex from 1 L culture of yeast cells. This amount of pure RF-C complex makes more detailed study on protein-protein interaction between RF-C complex, DNA polymerases and PCNA.Finally, we tried to express yeast DNA polymerases II and III complex using either yeast expression vectors or vaculovirus vector. However, so far we were not able to obtain any system expressing DNA polymerase complex. The main reason why we could not obtain any system that expresses yeast DNA polymerase complex was due to unstable recombinant vector DNA containing the gene encoding the DNA polymerase catalytic subunit in E.coli. Less

去年，我们成功地在Sf9昆虫细胞中使用液泡病毒载体表达了酵母酿酒酵母Cdc7/Dbf4蛋白激酶复合物，该复合物是启动酵母染色体DNA复制所必需的。今年，我们尝试开发一种新的、简单的纯化方法，以Mcm2蛋白为激酶底物，从感染含有CDC7和DBF4基因的重组病毒的Sf9细胞中纯化Cdc7/Dbf4蛋白复合物。使用 MonoQ、羟基磷灰石和肝素琼脂糖柱，我们能够纯化蛋白质复合物。使用纯化的 Cdc7/Dbf4 蛋白激酶复合物，我们发现该激酶不仅磷酸化酿酒酵母 Mcm2 蛋白，还磷酸化 Mcm3、Mcm4、Mcm6 和 Dbf4 蛋白。然而，在大肠杆菌中表达和纯化的 Mcm 蛋白并不是激酶的良好底物，这表明激酶更喜欢部分磷酸化的蛋白。使用含有 Gal 1 和 Gal10 启动子的酵母表达载体，我们能够表达由五个不同亚基（Rfc1p (Cdc44p)、Rfc2p、Rfc3p、 酵母中的 Rfc4p 和 Rfc5p)。在存在半乳糖的情况下，酵母细胞表达的 RF-C 活性比不存在半乳糖的情况至少高 50 倍。由于PCNA刺激RF-C复合物的ATP酶活性，因此RF-C复合物应与PCNA紧密相互作用。基于此，我们制作了PCNA缀合的琼脂糖柱。使用该柱，我们能够从 1 L 酵母细胞培养物中获得超过 mg 的高度纯化的 RF-C 复合物。这种量的纯RF-C复合物使得对RF-C复合物、DNA聚合酶和PCNA之间的蛋白质-蛋白质相互作用进行更详细的研究。最后，我们尝试使用酵母表达载体或空洞病毒载体表达酵母DNA聚合酶II和III复合物。然而，到目前为止我们还没有获得任何表达DNA聚合酶复合物的系统。我们无法获得表达酵母DNA聚合酶复合物的系统的主要原因是含有编码大肠杆菌DNA聚合酶催化亚基的基因的重组载体DNA不稳定。较少的

项目成果

Noskov, V.: "The RFC2 gene encoding the third largest subunit of the RF-C complex is required for chromosomal DNA replication and cell cycle checkpoint in the yeast Saccharomyces cerevisiae." Mol. Cell Biol.17 (in press). (1997)

Noskov, V.：“编码 RF-C 复合体第三大亚基的 RFC2 基因是酿酒酵母中染色体 DNA 复制和细胞周期检查点所必需的。”

Sugimoto, K.: "Rfc5, a small subunit of replicaton factor C complex, couples DNA replication and mitosis in budding yeast." Proc. Natl. Acad. Sci., USA. 93. 7048-7052 (1996)

Sugimoto, K.：“Rfc5 是复制因子 C 复合物的一个小亚基，在芽殖酵母中耦合 DNA 复制和有丝分裂。”

Hashimoto, K.: "The second subunit of DNA polymerse III (δ)is encoded by the HYS2 gene in Saccharomyces cerevisiae." J. Biol. Chem.272 (in press). (1997)

Hashimoto, K.：“DNA 聚合酶 III (δ) 的第二个亚基由酿酒酵母中的 HYS2 基因编码。”J. Biol.272（出版中）。

Hashimoto, K., Ohara, T., Maki, S., and Sugino, A.: "The second subunit of DNA polymerase III (delta) is encoded by the HYS2 gene in Saccharomyces cerevisiae." J.Biol.Chem.(in press). (1997)

Hashimoto, K.、Ohara, T.、Maki, S. 和 Sugino, A.：“DNA 聚合酶 III (δ) 的第二个亚基由酿酒酵母中的 HYS2 基因编码。”

Sugimoto,K.: "Rfc5,a small subunit of replication factor C complex,couples DNA replication and mitosis in budding yeast" Proc.Natl.Acad.Sci.,USA. 93. 7048-7052 (1996)

Sugimoto,K.：“Rfc5，复制因子 C 复合体的一个小亚基，在芽殖酵母中耦合 DNA 复制和有丝分裂”Proc.Natl.Acad.Sci.，美国。