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Publisher Summary This chapter discusses the construction of protein fragment complementation libraries using incremental truncation. Many proteins can have their peptide backbone cut by proteolytic or genetic means, yet the two fragments can associate to make an active heterodimer. This monomer-to-heterodimer conversion is referred to as protein fragment complementation (PFC). Such complementation is the reverse of evolutionary processes in which domains are recruited and fused at the genetic level. Incremental truncation, a method for rapidly generating a DNA library of every 1 base pair deletion of a gene or gene fragment, can be used to evaluate protein fragment complementation space. Plasmid pDIM-N2 is used for making libraries of N-terminal fragments, whereas pDIM-C8 is used for making libraries of C-terminal fragments. Each plasmid contains a different antibiotic resistance marker: ampicillin for pDIM-N2 and chloramphenicol for pDIM-C8. Each also has a different origin of replication so that both plasmids may be stably maintained in the same bacterial cell. The recovering and storing the individual truncation libraries is also elaborated. |
