Endolysin-Based Autolytic E. coli System for Facile Recovery of Recombinant Proteins

Recovery of recombinant proteins from the Escherichia coli cytoplasm depends on cell disruption by mechanical, chemical, and/or enzymatic methods, which usually cause incomplete cell breakage or protein denaturation. Controllable autolytic E. coli strains have been designed to facilitate the purification of recombinant proteins; however, these strains suffer from low recovery yield, slow cell lysis, or extensive strain engineering. Herein, we report an improved, highly efficient programmable autolytic E. coli platform, in which cell lysis is initiated upon the induced expression of T4 lysozyme with N-terminal fusion of a cell-penetrating peptide. Through the engineering of the peptide sequence and copy number, and by incorporating the fusion lytic gene into the E. coli genome, more than 99.97% of cells could be lysed within 30 min of induction regardless of cell age. We further tested the expression and release of a recombinant enzyme lysostaphin (Lst) and demonstrated that 4 h induction of the lytic gene after 3 h of Lst expression resulted in 98.97% cell lysis. Lst obtained from this system had the same yield, yet 1.63-fold higher activity, compared with that obtained from cells lysed by freeze-thawing and sonication. This autolytic platform shows potential for use in large-scale microbial production of proteins and other biopolymers.