Introduction of an aliphatic ketone into recombinant proteins in a bacterial strain that overexpresses an editing-impaired leucyl-tRNA synthetase

Abstract

A leucine analog containing a ketone has been incorporated into proteins in E. coli. Only E. coli strains overexpressing an editing-deficient leucyl-tRNA synthetase were capable of synthesizing proteins with the aliphatic ketone amino acid. Modification of ketone-containing proteins under mild conditions has been demonstrated.

Figure 1

Incorporation of 2 and 3 into target protein A1. A) Coomassie Brilliant Blue-stained SDS-PAGE gels of whole cell lysates after expressions in M9 –LIVM medium. Lanes i–iv: expression in host LAM1000/pA1EL/pREP4. Lanes v–viii: expression in host LAM1000/pA1T252Y/pREP4. Lanes i and v: induction without 1; lanes ii and vi: Induction with 1 at 40 mg L⁻¹; lanes iii and vii: induction with 2 at 100 mg L⁻¹; lanes iv and viii: induction with 3 at 200 mg L⁻¹. All concentrations are of the l-isomer. B) MALDI-MS of protein shown in lane viii. The single peak (found: 8307 Da; calculated: 8307 Da) confirms the identity of the protein.

Figure 2

Chemoselective modification of the ketone moiety in target protein A1. A)–C) Mass spectra of the tryptic fragment LKNEIEDLKAEIGDLNNTSGIR derived from A1; A) 1-A1 (found: 2442 Da; calculated: 2442 Da); B) 2-A1 (found: 2436 Da; calculated: 2436 Da); C) 3-A1 (found: 2442 Da; calculated: 2442 Da). Before hydroxylamine treatment, the mass of 3-A1 is nearly identical to that of 1-A1. After treatment with hydroxylamine (1 mm) in PBS (pH 6.5) for two hours at room temperature, mass peaks separated by 15 mass units were observed, corresponding to the conversion from ketone to oxime. Fragments shown in A) and B) were unmodified after treatment. D) Biotin-specific western blot analyses of 1-A1 and 3-A1 treated with biotin-hydrazide. Lane i: 1-A1 before treatment; ii: 3-A1 before treatment; iii: 1-A1 after treatment; iv: 3-A1 after treatment.

Figure 3

SDS-PAGE analysis of protein expression in cells overexpressing editing mutants of LeuRS. Expression in M9 –LIVM medium supplemented with 1, 3, or 4, was performed with E. coli cells overexpressing LeuRS containing Y, I, V, or T at position 252. After expression, whole cell lysates were subjected to SDS-PAGE, and proteins were visualized with Coomassie Brilliant Blue stain.

Scheme 1

Amino acids used in this study. 1: leucine; 2: didehydroleucine; 3: oxonorvaline; 4: norvaline.