The expression of recombinant proteins in E. coli followed by purification via affinity chromatography is an extremely popular strategy for obtaining relatively pure preparations of proteins for study. Unfortunately, the very first step in gaining access to these recombinant proteins, i.e. breaking open the bacterial cells, is often a dicey proposition. While many protocols favor the sonication route, this method can be very hard to control and essentially impossible to reproduce consistently. Oversonication, with the resultant foaming and potential damage to delicate proteins, or undersonication, with the ensuing inefficient bacterial lysis, are each surprisingly easy to accomplish. The lack of availability of a sonicator will necessitate the use of one of the many detergent-based protocols available. These usually involve some combination of lysozyme, Dnase, and Rnase. Regrettably, these methods never seem to work quite as efficiently as the written protocol suggests.
In an effort to satisfy the need for an easy, dependable bacterial cell lysis protocol, many vendors offer premixed solutions designed to crack open bacteria with minimal fuss. I recently tried the FastBreak Cell Lysis Reagent from Promega and found it to be quite effective and very easy. The FastBreak solution is a single-reagent system that can be supplemented with lysozyme and/or nucleases as needed. The reagent is supplied as a 10x solution that is added directly to the E. coli culture; no pelleting of the bacteria is needed. Although this feature makes the reagent very easy and economical to use on small cultures, it means that you will need 100 ml of the reagent ($150) to lyse a liter culture of bacteria.
I tested the FastBreak Cell Lysis Reagent on 1 ml aliquots of TG-1 E. coli grown to stationary phase. I supplemented the reagent with Dnase I to a final concentration of 10 µg/ml and then tested its ability to lyse the cells in the presence or absence of egg white lysozyme at 200 µg/ml. After a 15 min incubation with slow rotation at room temperature, I pelleted the unlysed cells by centrifugation for 10 min at 10,000 x g. An increased lysis efficiency was evident as the cell pellet from the lysozyme-containing condition was very small indeed and approximately one-third the size of the pellet from the condition without the enzyme. Measuring the absorbance of the supernantants at 280 nm (after correcting for the added protein in the lysozyme condition with an equivalent amount by weight of BSA) also showed an increase in the amount of protein released from the cells by a factor of approximately two. The absence of viscosity in the supernatants from both conditions suggested that the Dnase I was also effective in this reagent.
In summary, the FastBreak Cell Lysis Reagent from Promega is very easy to use and appears to be reasonably effective at lysing E. coli. The efficiency of the reagent can be markedly enhanced with lysozyme supplementation. The recommended protocol for lysis, i.e. adding it directly to bacterial cultures, seems to be more suited to small preps or screening clones for protein expression since using it to lyse large cultures can be somewhat expensive. Furthermore, the large volume of lysate solution, which is the same volume as the whole culture plus 10%, may be prohibitively large to use for some downstream applications. It could be that the reagent may be effective with pelleted cells under the proper conditions but I haven’t tried this yet.
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center