Journal of Microbiology

Research Support, Non-U.S. Gov'ts

Bacteria-Based In Vivo Peptide Library Screening Using Biopanning Approach: Ji-Hyeon Choi , Sang-Hyun Park; J. Microbiol. 2011;49(5):847-851. Published online November 9, 2011; DOI: https://doi.org/10.1007/s12275-011-1405-6

Abstract: Traditionally, library screening has been performed to identify biologically active agents including small molecules or peptides that inhibit target proteins or molecules with therapeutic interests. Due to its chemical nature, library screening is usually performed under in vitro environments using purified proteins and molecules. However, active agents identified from in vitro screenings often fail to exhibit biological activities in cells. To overcome this inherent limitation, we have developed an in vivo peptide library screening system that allows for the identification of dissociative inhibitors of protein interactions of interest. The screening is based on the reconstitution of the cI repressor from bacteriophage lambda with high-density expression peptide library and is entirely performed in bacteria cells. Furthermore, to enhance the efficacy and sensitivity of the screening, a multiple-round biopanning approach was employed for amplification and enrichment of positive peptides. Overall, this in vivo screening should provide a fast and efficient tool for identification of biologically active peptide molecules against target protein assembly.

Development of a Simple Cell Lysis Method for Recombinant DNA Using Bacteriophage Lambda Lysis Genes: Boyun Jang , Yuna Jung , Dongbin Lim; J. Microbiol. 2007;45(6):593-596.; DOI: https://doi.org/2602 [pii]

Abstract: 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.

Secondary Structure Analysis of Amino Terminal Domain in Phage Lambda Integrase: Yu, Jeong A , Nam, Chan Eun , Cho, Eun Hee; J. Microbiol. 1998;36(4):266-272.

Abstract: The amino-terminal domain of bacteriophage λ integrase recognizes specific DNA sequences called arm-type sites. To study the structural and functional relationships of the integras armtype DNA binding domains were confirmed by gel mobility-shift assay. The polypeptides were subjected to circular dichroism spectroscopy to estimate the amount of secondary structures they contain Based upon analyses of circular dichroism spectra and comparison with predicted secondary structural compositions, it was estimated that the amino terminal domain of integrase in an aqueous solution was composed of a little α-helical region. The helical content increased with an increasing amount of ethanol, an α-helix inducer. This indicates that its conformation can be changed to a form with higher content of α-helical structure under a certain condition.

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