Circular permutation of RNase T1 through PCR based site-directed mutagenesis
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Current methods for creating circularly permuted variants through alterations in the genetic sequence of a protein are laborious and involve a large number of recombinant DNA manipulations. This chapter discusses the utility of a general PCR approach for the creation of circularly permuted proteins through the initial characterization of cp35SL, and discusses the construction of a circularly permuted variant of ribonuclease Tl (RNase Tl). The native RNase Tl, (C2A, ClOA), and cp35Sl proteins are characterized by specific activity, polyacrylamide electrophoresis (SDS and native), thermodynamic stability, matrix-assisted laser desorption ionization (MALDI) mass spectrometry, and amino-terminal sequencing. The specific activity and thermodynamic stability of the (C2A, ClOA) mutant confirm that the Cys-2 to Cys-10 disulfide bond imparts thermodynamic stability but has little effect on catalytic activity. Hence this mutant was selected as the starting point for constructing a circularly permuted form of RNase-Tl, such that as short a linker as possible could be used to bridge the original N- and C-termini. The activity and stability of the circularly permuted variant indicate that, it adopts an overall tertiary fold very similar to that of the native protein. However, the real effect, however, may be more evident in the kinetics of the specific folding pathway. 1995, Elsevier Inc.
