title: Efficient expression and purification of human replication fork-stabilizing factor, Claspin, from mammalian cells: DNA-binding activity and novel protein interactions.
abstract: Purification of recombinant proteins of a large size often poses problems of instability or low expression in bacterial or insect cells. Here, we established a method for a high-level expression of large-sized recombinant proteins in mammalian cells and subsequent purification of the full-length proteins. We applied this method to express human Claspin and Tim-Tipin complex, which play important roles in replication checkpoint responses as fork-stabilizing factors, and successfully purified them in functional forms in amount sufficient for enzymatic characterization. Purified Claspin behaves as a monomer and binds preferentially to fork-like DNA. Over-expression of tagged Claspin in mammalian cells facilitated the detection of its interacting factors. Claspin interacts with many factors involved in checkpoint regulation and replication fork machinery, including ATR, ATM, Chk1, Tim, MCM4, MCM10, Cdc45, DNA polymerases α, δ, ε and Cdc7 kinase. We will discuss the potential implication of these findings in architecture of replication fork. We will also discuss the advantage of this system for purification and characterization of those proteins that are large and have been difficult to deal with.
authors.affiliation: Genome Dynamics Project, Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo 156-8506, Japan.
authors.forename: Syuzi
authors.initials: S
authors.lastname: Uno
authors.forename: Hisao
authors.initials: H
authors.lastname: Masai
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D011485:Protein Binding / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C488158:CLSPN protein, human; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D011994:Recombinant Proteins