[논문]Bacterial <tex>${\\beta}$</tex>-Lactamase Fragment Complementation Strategy Can Be Used as a Method for Identifying Interacting Protein Pairs

Park, Jong-Hwa; Back, Jung-Ho; Hahm, Soo-Hyun; Shim, Hye-Young; Park, Min-Ju; Ko, Sung-Il; Han, Ye-Sun

Bacterial ${\\beta}$-Lactamase Fragment Complementation Strategy Can Be Used as a Method for Identifying Interacting Protein Pairs 원문보기

Journal of microbiology and biotechnology, v.17 no.10, 2007년, pp.1607 - 1615

Park, Jong-Hwa (Department of Advanced Technology Fusion and Bio-Molecular Informatics Center, Konkuk University) , Back, Jung-Ho (Department of Advanced Technology Fusion and Bio-Molecular Informatics Center, Konkuk University) , Hahm, Soo-Hyun (Department of Advanced Technology Fusion and Bio-Molecular Informatics Center, Konkuk University) , Shim, Hye-Young (Department of Advanced Technology Fusion and Bio-Molecular Informatics Center, Konkuk University) , Park, Min-Ju (Department of Advanced Technology Fusion and Bio-Molecular Informatics Center, Konkuk University) , Ko, Sung-Il (Department of Advanced Technology Fusion and Bio-Molecular Informatics Center, Konkuk University) , Han, Ye-Sun (Department of Advanced Technology Fusion and Bio-Molecular Informatics Center, Konkuk University)

Abstract ▼ AI-Helper

We investigated the applicability of the TEM-l ${\beta}$-lactamase fragment complementation (BFC) system to develop a strategy for the screening of protein-protein interactions in bacteria. A BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD) was generated. The hFADD-hFasDD interaction was verified by cell survivability in ampicillin-containing medium and the colorimetric change of nitrocefin. It was also confirmed by His pull-down assay using cell lysates obtained in selection steps. A coiled-coil helix coiled-coil domain-containing protein 5 (CHCH5) was identified as an interacting protein of human uracil DNA glycosylase (hUNG) from the bacterial BFC cDNA library strategy. The interaction between hUNG and CHCH5 was further confirmed with immunoprecipitation using a mammalian expression system. CHCH5 enhanced the DNA glycosylase activity of hUNG to remove uracil from DNA duplexes containing a U/G mismatch pair. These results suggest that the bacterial BFC cDNA library strategy can be effectively used to identify interacting protein pairs.

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가설 설정

Cell lysates were collected and the expression of hUNG and CHCH5 was confirmed by Western blot analysis using anti-c-myc or anti-flag antibodies. B. Total cell lysates were collected from Hek293 cells transfected with different sets of plasmids. After coimmunoprecipitation using anti-c-myc antibody, elution samples were resolved by 12% polyaciylamide gel and analyzed by Western blot analysis using anti-flag or anti-c-myc antibodies.
In this report, we investigated the applicability of the TEM-1 BFC system in identifying interacting proteins in Escherichia coli cells. To accomplish this, we constructed the bait and prey plasmids of the BFC system containing a human Fas-associated death domain (hFADD) and human Fas death domain (hFasDD), which have been known to interact with each other [6], and analyzed p-lactamase activities by the complementation resulting from the hFADD-hFasDD interaction.

제안 방법

Purified hUNG fractions (50, 100, 200 ng) were reacted with 1 pmole of 5'-end labeled U/G mismatch 32 mer duplexes for 1 h at 37℃. The cleavage products were analyzed by 18% denaturing polyacrylamide gel electrophoresis and a BAS2000 image analyzer. A protein fraction (200 ng) purified from Hek293 cells transfected with pCMV tag3A by the same methods as hUNG purification was reacted as a negative control (lane 3).
6). To investigate the effect of CHCH₅ protein on the DNA 읺ycosylase activity of hUNQ CHCH₅ and hUNG were transiently expressed in Hek293 cells and purified by immunoaffinity batch chromatography. DNA glycosylase activities of purified hUNG increased in a dose-dependent manner (Fig.
Two plasmid vectors, pSLBLC-c-myc-hFADD andpSBLNL- hFasDD-His, were designed and constructed to complement an enzymatic activity of p-lactamase by T7 promoter driven coexpression of an interacting protein pair, hFADD and hFasDD. The a-197 and cd-198 fragments of p-lactamase were fused through a triple pentapeptide (GGGGS) linker to the N-terminal of hFasDD and the C-terminal of hFADD, respectively (Fig.

성능/효과

In conclusion, we established the E. coli BFC strategy and investigated the applicable possibilities to identify interacting proteins from cDNA libraries fosed to the complementary P-lactamase fiagment CHCH₅ was identified as a candidate to interact with hUNGl though this strategy, and hUNG DNA 이ycosylase activity was dramatically increase by CHCH₅. These results mean that E.

후속연구

Since CHCH₅ was identified to interact with 78- 304 amino acid residues of hUNQ which are conserved in hUNGl and hUNG2, CHCH₅ is also expected to be involved in removing uracil residues by hUNG2 in a post replicative process. Although further investigations are necessary to establish the real mechanism and function of hUNG-CHCH₅ interaction, these results stron읺y suggest that CHCH₅ interacts with hUNG and participates in the BER pathway to remove misincorporated uracil residues via enhancing hUNG DNA glycosylase activity.

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