研究者詳細 - 服部　高子

Production of recombinant CCN2 protein in Escherichia coli 国際誌

Eriko Aoyama, Takako Hattori, Satoshi Kubota, Masaharu Takigawa

Methods in Molecular Biology 1489 77 - 84 2017年

記述言語：英語出版者・発行元：Humana Press Inc.

Recombinant proteins are important tools for understanding molecular functions in vitro. Recent progress in the generation of recombinant proteins is amazing. However, when we plan to produce them, we should choose the best method according to the nature and the use of the target recombinant protein. Degradation and mis-folding are major problems in producing active recombinant CCN2. The method shown in this chapter describes the appropriate conditions under which we can produce CCN2 and its truncated fragments in Escherichia coli.

DOI： 10.1007/978-1-4939-6430-7_8

Protocols for screening for binding partners of CCN proteins: Yeast two-hybrid system 国際誌

Mitsuhiro Hoshijima, Takako Hattori, Masaharu Takigawa

Methods in Molecular Biology 1489 145 - 154 2017年

記述言語：英語出版者・発行元：Humana Press Inc.

Yeast two-hybrid screening is a powerful method to identify proteins that interact with a protein of interest. CCN2 consists of four domains, and identification of new proteins that bind to individual domains of CCN2 may reveal a variety of CCN2 functions. To identify CCN2-interactive proteins that regulate CCN2 activity, we carried out GAL4-based yeast two-hybrid screening with a cDNA library derived from a chondrocytic cell line, HCS-2/8, with CCN2 cDNA used as a bait. In this chapter, we describe our methods for screening for CCN2 binding partners by this system in detail. This protocol may be applied to other CCN proteins as well.

DOI： 10.1007/978-1-4939-6430-7_15

Generation and analysis of cartilage-specific CCN2 overexpression in transgenic mice 国際誌

Takako Hattori, Shinsuke Itoh, Masaharu Takigawa

Methods in Molecular Biology 1489 391 - 403 2017年

記述言語：英語出版者・発行元：Humana Press Inc.

Recent progress in gene-editing technology has provided a strong impact for improved our understanding of molecular functions in living organisms. Here we describe our method to generate transgene-overexpressing mouse models, which method involves the use of tissue-specific promoters for analyzing a certain molecule (s) in special tissues. The protocol described in this chapter uses the Col2a1 promoter-enhancer, which is known for driving specific and strong transgene expression in cartilage and is based on several of our studies showing a positive role of the connective tissue growth factor (CCN2) in cartilage-bone development and maintenance of articular cartilage. These mice show strongly accelerated endochondral ossification resulting in enhanced bone elongation, as well as resistance to age-related articular degeneration. This protocol also describes how to analyze the molecular mechanisms of these phenomena by use of chondrocytes isolated from CCN2-overexpressing cartilage.

DOI： 10.1007/978-1-4939-6430-7_32