Novel method to rapidly and efficiently lyse Escherichia coli for the isolation of recombinant protein.

Rapid and high-throughput protein purification methods are required to explore structure and function of several uncharacterized proteins. Isolation of recombinant protein expressed in Escherichia coli strain BL21 (DE3) depends largely on the efficient and speedy bacterial cell lysis, which is considered as the bottleneck during protein purification. Cells are usually lysed by either sonication or high pressure homogenization, both of which are slow, require special equipment, lead to heat generation, and may result in loss of protein's biological activity. We report here a novel method to lyse E. coli, which is rapid, and results in high yield of isolated protein. Here, we have carried out intracellular expression of lysozyme domain (LD) of mycobacteriophage D29 endolysin. LD remains non-toxic until chloroform is added into the culture medium that permeabilizes bacterial cell membrane and allows the diffusion of LD to the peptidoglycan layer causing latter's degradation ensuing cell lysis. Our method efficiently lyses E. coli in short duration. As a proof-of-concept, we demonstrate large scale isolation and purification of α subunit of E. coli RNA polymerase and GFP, when they are co-expressed with LD. We believe that our method will be adopted easily in high-throughput as well as large scale protein isolation experiments.