title: The expression and crystallization of Cry65Aa require two C-termini, revealing a novel evolutionary strategy of Bacillus thuringiensis Cry proteins.
abstract: The insecticidal crystal protein (Cry) genes of Bacillus thuringiensis are a key gene resource for generating transgenic crops with pest resistance. However, many cry genes cannot be expressed or form crystals in mother cells. Here, we report a novel Cry protein gene, cry65Aa1, which exists in an operon that contains a downstream gene encoding a hypothetical protein ORF2. We demonstrated that ORF2 is required for Cry65Aa1 expression and crystallization by function as a C-terminal crystallization domain. The orf2 sequence is also required for Cry65Aa expression, because orf2 transcripts have a stabilizing effect on cry65Aa1 transcripts. Furthermore, we found that the crystallization of Cry65Aa1 required the Cry65Aa1 C-terminus in addition to ORF2 or a typical Cry protein C-terminal region. Finally, we showed that Cry65Aa1 has a selective cytotoxic effect on MDA-MB231 cancer cells. This report is the first description of a 130-kDa mass range Cry protein requiring two C-termini for crystallization. Our findings reveal a novel evolutionary strategy of Cry proteins and provide an explanation for the existence of Cry protein genes that cannot form crystals in B. thuringiensis. This study also provides a potential framework for isolating novel cry genes from "no crystal" B. thuringiensis strains.
authors: State Key Laboratory of Agricultural Microbiology, College of life science and technology, Huazhong Agricultural University, Wuhan 430070, Hubei, People's Republic of China.
authors: Dong-hai
authors: DH
authors: Peng
authors: State Key Laboratory of Agricultural Microbiology, College of life science and technology, Huazhong Agricultural University, Wuhan 430070, Hubei, People's Republic of China.
authors: Cui-yun
authors: CY
authors: Pang
authors: State Key Laboratory of Agricultural Microbiology, College of life science and technology, Huazhong Agricultural University, Wuhan 430070, Hubei, People's Republic of China.
authors: Han
authors: H
authors: Wu
authors: State Key Laboratory of Agricultural Microbiology, College of life science and technology, Huazhong Agricultural University, Wuhan 430070, Hubei, People's Republic of China.
authors: Qiong
authors: Q
authors: Huang
authors: State Key Laboratory of Agricultural Microbiology, College of life science and technology, Huazhong Agricultural University, Wuhan 430070, Hubei, People's Republic of China.
authors: Jin-shui
authors: JS
authors: Zheng
authors: State Key Laboratory of Agricultural Microbiology, College of life science and technology, Huazhong Agricultural University, Wuhan 430070, Hubei, People's Republic of China.
authors: Ming
authors: M
authors: Sun
mesh_terms: D001413:Bacillus thuringiensis / Q000502:physiology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000633:toxicity; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D016000:Cluster Analysis; D003460:Crystallization; D019143:Evolution, Molecular; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D009690:Nucleic Acid Conformation; D054730:Protein Interaction Domains and Motifs; D020871:RNA Stability; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D012333:RNA, Messenger
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