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

在过去的一年里，我们成功地利用空泡病毒载体在Sf9昆虫细胞中表达了酵母染色体DNA复制所需的酵母CDC7/Dbf4蛋白激酶复合体。在这一年里，我们试图建立一种新的、简单的从感染CDC7和DBF4基因的重组病毒感染的Sf9细胞中纯化CDC7/Dbf4蛋白复合体的方法，该方法以Mcm2蛋白为底物。使用MonoQ、羟基磷灰石和肝素-Sepharose柱，我们能够纯化蛋白质复合体。利用纯化的CDC7/Dbf4蛋白激酶复合体，我们发现该蛋白不仅能磷酸化酿酒酵母Mcm2蛋白，还能磷酸化MCM3、Mcm4、Mcm6和Dbf4蛋白。然而，在大肠杆菌中表达和纯化的MCM蛋白不是该蛋白的良好底物，这表明该蛋白更倾向于部分磷酸化的蛋白。利用含有Gal1和Gal10启动子的酵母表达载体，我们能够表达酵母R…酵母中更多的F-C复合体由5个不同的亚基(Rfc1p(CDc44p)、Rfc2p、Rfc3p、Rfc4p和Rfc5p)组成。在有半乳糖存在的情况下，酵母细胞表达的RF-C活性至少是在没有半乳糖的情况下的50倍。由于增殖细胞核抗原能刺激RF-C复合体的ATPase活性，因此，RF-C复合体应该与PCNA紧密结合，在此基础上，我们制备了一种结合了PCNA的琼脂糖柱。使用此柱，我们能够从1个L酵母细胞培养物中获得高纯度的RF-C复合体。这一数量的纯RF-C复合体对RF-C复合体、DNA聚合酶和PCNA之间的蛋白质-蛋白质相互作用进行了更详细的研究。最后，我们尝试用酵母表达载体和空泡病毒载体表达酵母DNA聚合酶II和III复合体。然而，到目前为止，我们还没有得到任何表达DNA聚合酶复合体的系统。我们无法获得任何酵母DNA聚合酶复合体表达系统的主要原因是重组载体DNA在大肠杆菌中含有编码DNA聚合酶催化亚基的基因不稳定。较少

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