Purification of replication factors using insect and mammalian cell expression systems.
Purification of factors for DNA replication in an amount sufficient for detailed biochemical characterization is essential to elucidating its mechanisms. Insect cell expression systems are commonly used for purification of the factors proven to be difficult to deal with in bacteria. We describe first the detailed protocols for purification of mammalian Mcm complexes including the Mcm2/3/4/5/6/7 heterohexamer expressed in insect cells. We then describe a convenient and economical system in which large-sized proteins and multi-factor complexes can be transiently overexpressed in human 293T cells and be rapidly purified in a large quantity. We describe various expression vectors and detailed methods for transfection and purification of various replication factors which have been difficult to obtain in a sufficient amount in other systems. Availability of efficient methods to overproduce and purify the proteins that have been challenging would facilitate the enzymatic analyses of the processes of DNA replication.
Department of Genome Medicine, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
Shuji
S
Uno
Zhiying
Z
You
Hisao
H
Masai
D048868:Adaptor Proteins, Signal Transducing / Q000096:biosynthesis / Q000302:isolation & purification; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D005822:Genetic Vectors; D057809:HEK293 Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D061987:Sf9 Cells; D014162:Transfection
D048868:Adaptor Proteins, Signal Transducing; C488158:CLSPN protein, human; D004268:DNA-Binding Proteins; D046912:Multiprotein Complexes; D011994:Recombinant Proteins