Notes & Tips Analysis of protein interactions with two-hybrid system in cultured insect cells Hiroaki Mon a , Ryohei Sugahara a , Sun-Mee Hong a , Jae-Man Lee a , Yusuke Kamachi b , Yutaka Kawaguchi a , Takahiro Kusakabe a, * a Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Fukuoka 812-8581, Japan b Graduate School of Frontier Biosciences, Developmental Biology Group, Osaka University, Osaka 565-0871, Japan article info Article history: Received 21 April 2009 Available online 27 May 2009 abstract Many biological processes are usually coupled to the formation of protein complexes. The yeast two- hybrid system is a powerful tool for analyzing protein–protein interactions. Different patterns of protein modifications, such as glycosylation, phosphorylation, and acetylation, may affect the ability of proteins to interact. In this study, we developed the two-hybrid system that can be used in insect cells. To validate the insect two-hybrid (I2H) system, we analyzed and confirmed the known oligomer or dimer formation of silkworm Rad51 or RPA2–RPA3, respectively. The results established the feasibility of the I2H system for efficient analysis of protein interaction under conditions that closely reflect the normal physiological environment. Ó 2009 Elsevier Inc. All rights reserved. Dynamic networks of protein–protein interactions regulate numerous cellular processes. The analytical methods that permit revealing how each protein interacts with its partners under native conditions provide insight into the cellular functions of proteins. Several approaches have so far been reported to study protein–pro- tein interactions in living cells, and each method has its own advantages and limitations [1]. The yeast two-hybrid system is widely used to detect and identify protein–protein interactions [2]. However, the yeast two-hybrid system is well known to pro- duce false-positive and false-negative interactions. Furthermore, interactions that require some posttranslational modifications are not observed in yeast. Generating a system to study protein–pro- tein interactions using an insect host cell would provide an alter- native method for capturing protein–protein interactions that cannot be studied in yeast. Here we describe the construction of an insect two-hybrid (I2H) 1 system in which the two proteins of interest are fused to the yeast GAL4 DNA binding domain and mouse nuclear factor-kappa B (NF-jB) transcriptional activation domain, respectively [2]. An interaction between the two proteins results in the expression of the firefly luciferase reporter gene, which enables detecting weak protein interactions. The I2H system is similar to the yeast two-hybrid system based on the fact that transcription factors are composed of two functional domains: a DNA binding domain (DBD) and an activation domain (AD). The gene encoding the protein of interest (X) is fused to a DBD, and the gene encoding the potential interacting partner (Y) is fused to an AD. If protein X interacts with protein Y, the AD is brought into close proximity to the DBD, resulting in the expression of the re- porter gene (Fig. 1A). If protein X does not interact with protein Y, the reporter gene does not express (Fig. 1B). To develop the I2H technol- ogy for the detection of protein–protein interactions in cultured in- sect cells, we have generated three vectors: pIE2–DBD, pIE2–AD, and pUAS–Luc. The first plasmid, pIE2–DBD, is a Gateway destination vector to clone a gene of interest in frame with the sequence encod- ing the GAL4 DBD. The second plasmid, pIE2–AD, is also a Gateway destination vector to clone a gene in frame with the sequence encod- ing the activation domain of mouse NF-jB (p65) (Fig. 1C). The OpIE2 promoter, the Gateway cassette with adjacent HA or FLAG tag, and the OpIE2 polyadenylation sequence were amplified by polymerase chain reaction (PCR) from pie2HW or pie2FW [3]. The reverse prim- ers used were as follows: HA, 5 0 -CAAACTAGT A TACCGGTGTC CGCCA TGAGC AGCG-3 0 ; FLAG, 5 0 -CAAACTAGT A TACCGGTGCT TGTCATCG TC ATCC-3 0 . An identical forward primer, 5 0 -acaagtttgtacaaaa aagctg-3 0 , was used for two PCR reactions. GAL4–DBD and mouse NF-jB transcriptional AD were amplified by PCR from pBacMC- S[A3-gal4-NF-jB] with the following sets of primers: DBD, 5 0 - CGCACTAGT A TGAAGCTACT GTCTTCTATC GAAC-3 0 and 5 0 -CGATAC AGTC AACTGTCTTT GACCTTTG-3 0 ; AD, 5 0 -TAACTAGT GA ATTCCC TTCT GGGCAAATCT CAAAC-3 0 and 5 0 -TTGGCCGCTG GAGCTGATCT GACTCAGCAG-3 0 [4]. The PCR products were ligated to create pIE2–DBD and pIE2–AD, respectively. All ligation reactions, includ- 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.05.033 * Corresponding author. Address: Laboratory of Silkworm Science, Kyushu University, Kyushu University Graduate School of Bioresource and Bioenvironmen- tal Sciences, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan. Fax: +81 92 642 2842. E-mail address: kusakabe@agr.kyushu-u.ac.jp (T. Kusakabe). 1 Abbreviations used: I2H, insect two-hybrid; NF-jB, nuclear factor-kappa B; DBD, DNA binding domain; AD, transcriptional activation domain; PCR, polymerase chain reaction; IE2, immediate–early promoter; UAS, GAL4 upstream activating sequences. Analytical Biochemistry 392 (2009) 180–182 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage: www.elsevier.com/locate/yabio