Biopolymers and cell. 2003; 19 (3): 274 - 280

Overexpression and purification of methionine aminopeptidase from Escherichia coli

I. Yu. Slavchenko, E. V. Boreyko, N. V. Vorobey, T. G. Gavrysh, E. N. Pehota, V. A. Kordyum

Methionine aminopeptidases (MAPs) play an important role in protein processing. In both eukaryotic and prokaryotic cells MAPs selectively remove methionine residue from the N-termini of a nascent polypeptide. However, an extra N-terminal methionyl residue frequently remains in heterologous proteins being produced in bacteria. In case of recombinant proteins destined for clinical application, it is utmost important that the final product has an N-terminal identical to that of a natural counterpart, as otherwise it may possess antigenic properties. An extra N-terminal methionine residue of a recombinant polypeptide may be removed in vitro by treatment with methionine aminopeptidase from E. coli. In the present work, an effective method of this enzyme production has been developed using an expression system based on the bacteriophage T7 polymerase. It was shown, that upon the cultivation of the MAP producer BL (pET-MAP) at 37 °C the target protein mainly accumulated in an insoluble form with a yield 17 % of the total cellular protein. However, if the producer cells are infected by phage λ, a lysate obtained contains MAP in a soluble form with higher yield compared with non infected cells. Optimization of the conditions such as cells density at the phage infection and temperature of the producer cultivation resulted in the target product yield exceeding 40 % of the soluble cell proteins, or 0,8 g/1. The recombinant protein has been purified by ion-exchange chromatography to homogeneity not less than 90 %.