Two-stage gene assembly/cloning of a member of the TspDTI subfamily of bifunctional restriction endonucleases, TthHB27I
Two-stage gene assembly/cloning of a member of the TspDTI subfamily of bifunctional restriction endonucleases, TthHB27I
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DOI：
10.1016/j.jbiotec.2014.11.030
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发表时间：
2015-01-20
影响因子：
4.1
通讯作者：
Skowron, Piotr M.
中科院分区：
文献类型：
--
作者：
Krefft, Daria;Zylicz-Stachula, Agnieszka;Skowron, Piotr M.
Therms sp。双功能型 IIS/IIG/IIC 限制性内切酶 (REase)-甲基转移酶 (MTase) 家族包括热稳定性 TaqII、TspGWI、TspDTI、TsoI、Tth111II/TthHB27I 酶以及许多假定的酶/开放阅读框 (ORF)。所有家族成员都有共同的特性，包括大蛋白大小（约 120 kDa）、氨基酸 (aa) 序列同源性、S-腺苷甲硫氨酸 (SAM) 的酶活性调节、相似的不对称同源 DNA 位点识别以及 1119 nt 距离处的切割。对酶氨基酸序列和结构域/基序组织的分析导致了进一步的Therms sp。家族分为 TspDTI 和 TspGWI 亚家族。后者在用其类似物 Sinefungin (SIN) 取代 SAM 后表现出前所未有的 DNA 识别变化现象，导致非常频繁的 DNA 裂解。我们报告了在大肠杆菌 (E. coli) 中的克隆，使用两阶段程序和推定的 tthHB27IRM 基因，通过嗜热嗜热菌 HB27 (T. thermophilus) 基因组的生物信息学分析检测到。 3366 个碱基对 (bp)/1121 个氨基酸长、高 GC 含量 ORF 的功能通过在大肠杆菌中的表达进行了实验验证。蛋白质特征证实了 TthHB27I 重新分类为 TspDTI 亚家族，这表现为 aa 序列/基序同源性和对 SIN 诱导的特异性转变不敏感。然而，SAM 和 SIN 都将 REase DNA 切割活性刺激了至少 16-32 倍； Therms sp 观察到最高。家庭。 TthHB27I 的可用性以及在反应中包含 SAM 或 SIN 的需要，以便将酶从“冬眠”状态转化为有效的 DNA 切割，在分子生物技术中具有实际意义，扩展了可用 REase 特异性的范围。 (C) 2014 Elsevier B.V. 保留所有权利。
The Therms sp. family of bifunctional type IIS/IIG/IIC restriction endonucleases (REase)-methyltransferases (MTase) comprises thermo-stable TaqII, TspGWI, TspDTI, TsoI, Tth111II/TthHB27I enzymes as well as a number of putative enzymes/open reading frames (ORFs). All of the family members share properties including a large protein size (ca. 120 kDa), amino acid (aa) sequence homologies, enzymatic activity modulation by S-adenosylmethionine (SAM), recognition of similar asymmetric cognate DNA sites and cleavage at a distance of 1119 nt. Analysis of the enzyme aa sequences and domain/motif organisation led to further Therms sp. family division into the TspDTI and TspGWI subfamilies. The latter exhibits an unprecedented phenomenon of DNA recognition change upon substitution of SAM by its analogue, sinefungin (SIN), towards a very frequent DNA cleavage. We report cloning in Escherichia coli (E. coli), using a two-stage procedure and a putative tthHB27IRM gene, detected by bioinformatics analysis of the Therms thermophilus HB27 (T. thermophilus) genome. The functionality of a 3366 base pair (bp)-/1121 aa-long, high GC content ORF was validated experimentally through the expression in E. coli. Protein features corroborated with the reclassification of TthHB27I into the TspDTI subfamily, which manifested in terms of aa-sequence/motif homologies and insensitivity to SIN-induced specificity shift. However, both SAM and SIN stimulated the REase DNA cleavage activity by at least 16-32 times; the highest was observed for the Therms sp. family. The availability of TthHB27I and the need to include SAM or SIN in the reaction in order to convert the enzyme from "hibernation" status to efficient DNA cleavage is of practical significance in molecular biotechnology, extending the palette of available REase specificities. (C) 2014 Elsevier B.V. All rights reserved.