Development of a simple cell lysis method for recombinant DNA using bacteriophage lambda lysis genes.
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.
Jang
Boyun
B
Department of Bioinformatics and Life Science, Soongsil University, Seoul, Republic of Korea.
Jung
Yuna
Y
Lim
Dongbin
D
D001433:Bacteriolysis; D010582:Bacteriophage lambda / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D004274:DNA, Recombinant / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D005615:Freezing; D050296:Microbial Viability / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D013010:Sonication; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D004274:DNA, Recombinant; D014764:Viral Proteins