Jang Boyun, Jung Yuna, Lim Dongbin
Department of Bioinformatics and Life Science, Soongsil University, Seoul, Republic of Korea.
J Microbiol. 2007 Dec;45(6):593-6.
In this study, we describe the development of a simple and efficient method for cell lysis via the insertion of a bacteriophage lambda lysis gene cluster into the pET22b expression vector in the following order; the T7 promoter, a gene for a target protein intended for production, Sam7 and R. This insertion of R and Sam7 into pET22b exerted no detrimental effects on cellular growth or the production of a target protein. The induction of the T7 promoter did not in itself result in the autolysis of cells in culture but the harvested cells were readily broken by freezing and thawing. We compared the efficiency of the cell lysis technique by freezing and thawing to that observed with sonication, and determined that both methods completely disintegrated the cells and released proteins into the solution. With our modification of pET22b, the lysis of cells became quite simple, efficient, and reliable. This strategy may prove useful for a broad variety of applications, particularly in experiments requiring extensive cell breakage, including library screening and culture condition exploration, in addition to protein purification.
在本研究中，我们描述了一种简单高效的细胞裂解方法的开发过程，即通过按以下顺序将噬菌体λ裂解基因簇插入pET22b表达载体中：T7启动子、用于生产目标蛋白的基因、Sam7和R。将R和Sam7插入pET22b对细胞生长或目标蛋白的产生没有不利影响。T7启动子的诱导本身不会导致培养细胞的自溶，但收获的细胞通过冻融很容易破碎。我们将冻融细胞裂解技术的效率与超声处理的效率进行了比较，确定这两种方法都能使细胞完全解体并将蛋白质释放到溶液中。通过对pET22b的改造，细胞裂解变得非常简单、高效且可靠。这种策略可能对广泛的应用有用，特别是在需要大量细胞破碎的实验中，包括文库筛选、培养条件探索以及蛋白质纯化。
