Circular permutation in the Ω-loop of TEM-1 β-lactamase results in improved activity and altered substrate specificity

Abstract

Generating diverse protein libraries that contain improved variants at a sufficiently high frequency is critical for improving the properties of proteins using directed evolution. Many studies have illustrated how random mutagenesis, cassette mutagenesis, DNA shuffling and similar approaches are effective diversity generating methods for directed evolution. Very few studies have explored random circular permutation, the intramolecular relocation of the N- and C-termini of a protein, as a diversity-generating step for directed evolution. We subjected a library of random circular permutations of TEM-1 β-lactamase to selections on increasing concentrations of a variety of β-lactam antibiotics including cefotaxime. We identified two circularly permuted variants that conferred elevated resistance to cefotaxime but decreased resistance to other antibiotics. These variants were circularly permuted in the Ω-loop proximal to the active site. Remarkably, one variant was circularly permuted such that the key catalytic residue Glu166 was located at the N-terminus of the mature protein.

Figure 1. Library construction.

(A) The previously described collection of circularly permuted bla genes was PCR-amplified using primers designed to anneal just outside the circularly permuted bla DNA. Both primers contained an appropriately spaced BsgI restriction site (cuts and the indicated dashed lines) such that treatment of the digested product to remove the two-base 3′ overhand would result in the circularly permuted bla library without any “scars” from the surrounding DNA. (B) Plasmid pC8BlaStop is derived from pDIM-C8 and contains appropriately place SapI and AflII sites such that fusion of the circularly permuted bla library can occur seamlessly to the bla signal sequence (blass) and a series of stop codons in all three reading frames (in bold).

Figure 2

Sites of circular permutation BLA based on DNA sequencing of (A) 25 randomly selected members from the naïve library, (B) 20 randomly selected members capable of growing on plates containing 16 µg/ml ampicillin, and (C) 10 randomly selected members capable of growing on plates containing 250 µg/ml ampicillin. The first amino acid in the mature wild-type BLA is 24 (since the signal sequence of amino acids 1–23 is removed) and the last amino acid is 286. The linker joining the N- and C-termini has the amino acid sequence DKS.