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Applied and Environmental Microbiology, August 1999, p. 3464-3469, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Activation Process of Dipteran-Specific Insecticidal Protein Produced by Bacillus thuringiensis subsp. israelensis

Masashi Yamagiwa,¹ Motoyuki Esaki,¹ Kanao Otake,¹ Manabu Inagaki,¹ Tohru Komano,² Teruo Amachi,¹ and Hiroshi Sakai^1,*

Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502,¹ and Department of Genetic Engineering, Kinki University, Wakayama 649-6493,² Japan

Received 4 January 1999/Accepted 6 June 1999

Dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Here we performed the in vitro processing of Cry4A and showed that the 130-kDa protoxin of Cry4A was processed into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60-kDa intermediate. The processing into these two fragments was also observed in vivo. To investigate functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60-kDa intermediate and the 20- and 45-kDa fragments were constructed. Neither the GST-20-kDa fusion protein (GST-20) nor the GST-45-kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST-60-kDa intermediate fusion protein (GST-60) exhibited significant toxicity. However, when the two fusion proteins GST-20 and GST-45 coexisted, significant toxicity was observed. The coprecipitation experiment demonstrated that the two fragments associated with each other. Therefore, it is strongly suggested that the two fragments formed an active complex of apparently 60 kDa. A mutant of the 60-kDa protein which was apparently resistant to the intramolecular cleavage with the midgut extract of C. pipiens larvae had toxicity slightly lower than that of GST-60.

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^* Corresponding author. Present address: Laboratory of Gene Engineering, Department of Bioscience and Biotechnology, Faculty of Engineering, Okayama University, Tsushima-Naka 3-1-1, Okayama-shi, Okayama 700-8530, Japan. Phone/fax: 81 86 251 8203. E-mail: sakahrsh{at}biotech.okayama-u.ac.jp.

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Applied and Environmental Microbiology, August 1999, p. 3464-3469, Vol. 65, No. 8
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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