Ls. Mullins et al., TRANSPOSITION OF PROTEIN SEQUENCES - CIRCULAR PERMUTATION OF RIBONUCLEASE-T1, Journal of the American Chemical Society, 116(13), 1994, pp. 5529-5533
A general procedure has been developed for the simple and rapid constr
uction of circularly permuted proteins. The polymerase chain reaction
(PCR) was utilized to amplify the gene for ribonuclease T1 (RNase T1)
in two separate pieces, and these fragments were recombined in reverse
order to create the circularly permuted gene. The circularly permuted
analog of RNase T1 was assembled by first removing the disulfide bond
between Cys-2 and Cys-10 through substitution with alanine residues t
o create the mutant (C2A, C10A). The original amino- and carboxyl-term
inal ends of (C2A, C10A) were then covalently linked with the peptide
Gly-Pro-Gly, and new termini were introduced between residues Gly-34 a
nd Ser-35. The bacterially expressed and circularly permuted variant o
f RNase T1, cp35S1, was 28% as active as the native enzyme in the cata
lytic hydrolysis of RNA, and thus the mutant protein must fold to a co
nformational state quite similar to that of the native enzyme. Amino a
cid sequence analysis and mass spectrometry have confirmed the primary
structure of the reconstructed protein. Thermodynamic stabilities at
pH 5.0 of wild-type RNase T1, (C2A, C10A), and cp35S1 proteins were fo
und to be 10.1, 6.4, and 4.2 kcal/mol, respectively.