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Bacterial Cell Lysis Buffer

Bacterial Cell Lysis Buffer

At NZYTech we intend to develop, manufacture and market solutions that simplify, accelerate and improve life sciences research. Thus, conscious of the pressures that befall on structural genomics and proteomics as a result of the dramatic increasing number of genome sequences available nowadays, we have recently developed a high-throughput (HTP) platform to efficiently clone and express a large amount of recombinant proteins that can be rapidly used in subsequent protocols for function and structure determination. When implementing a HTP platform for production of hundreds of proteins at the same time it is, obviously, necessary to modify the modus operandi of the cloning, expression and production protocols. So, the conventional cell lysis using mechanical procedures such as French Press or sonication, even adapted for HTP approaches, is not practical and economical and the solution is to use readyto-use chemicals for simultaneous bacterial cell walls disruption. Here, we would like to introduce an innovative reagent for the gentle disruption of Escherichia coli cell wall, generating a homogeneous cell-free extract. Even for laboratories studying a single protein target, such as designed proteins, the usage of this product is beneficial as the yield of extracted protein will be significantly improved besides it is less time-consuming than the mechanical lysis. NZY Bacterial Cell Lysis Buffer (Cat. No. MB1780) is a Tris-buffered formulation (pH 7.5) with lysozyme and DNase I provided separately for more convenient use. The addition of these enzymes allows a most efficient extraction, however for some over-expressed proteins and particular E. coli strains the addition of lysozyme may not be required. Furthermore, the presence of lysozyme and DNase I might interfere with some downstream applications, so in these situations the addition of these enzymes should be omitted. Additional components, such as protease inhibitors, salts, reducing agents and chelating agents (not provided), may be added to the lysate obtained using the NZY Bacterial Cell Lysis Buffer, depending on the particular application. The efficiency of the chemical lysis provided by NZY Bacterial Cell Lysis Buffer was compared to a mechanical procedure (sonication) in a test with 22 cultures containing recombinant Escherichia coli BL21(DE3) expressing different proteins from the anaerobic ruminal bacterium Ruminococcus flavefaciens (P1-P22). All E. coli cultures containing recombinant plasmids were cultured, in duplicate, in 5 mL of NZY Auto-Induction LB medium (NZYTech, Cat. No. MB179) supplemented with appropriate antibiotic. After growth and induction at 30 °C during 16h, cells were harvest at 5,000 ×g. Each cell-pelleted culture was lysed, in parallel, by sonication and chemical lysis provided by NZY Bacterial Cell Lysis Buffer. Recombinant proteins extracted using these two strategies were purified through Immobilized Metal Affinity 2 Chromatography (IMAC) and separated by SDS-PAGE. The levels of protein obtained were evaluated (Figure 1). In general, the data revealed that NZY Bacterial Cell Lysis Buffer extracts shows higher or similar levels of target protein when compared with sonication. A detailed analysis of data collected reveals that for only four proteins in test, sonication performed slightly better than the NZYTech’s protein extraction reagent. Besides the high levels of recombinant proteins obtained after cell lysis with the NZY Bacterial Cell Lysis Buffer, this procedure offers a rapid and less labor-intensive strategy than sonication, when purifying multiple proteins in parallel

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