Department of Molecular Biotechnology, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, Gdansk, Poland.
PLoS One. 2017 Oct 17;12(10):e0186633. doi: 10.1371/journal.pone.0186633. eCollection 2017.
Obtaining thermostable enzymes (thermozymes) is an important aspect of biotechnology. As thermophiles have adapted their genomes to high temperatures, their cloned genes' expression in mesophiles is problematic. This is mainly due to their high GC content, which leads to the formation of unfavorable secondary mRNA structures and codon usage in Escherichia coli (E. coli). RM.TthHB27I is a member of a family of bifunctional thermozymes, containing a restriction endonuclease (REase) and a methyltransferase (MTase) in a single polypeptide. Thermus thermophilus HB27 (T. thermophilus) produces low amounts of RM.TthHB27I with a unique DNA cleavage specificity. We have previously cloned the wild type (wt) gene into E. coli, which increased the production of RM.TthHB27I over 100-fold. However, its enzymatic activities were extremely low for an ORF expressed under a T7 promoter. We have designed and cloned a fully synthetic tthHB27IRM gene, using a modified 'codon randomization' strategy. Codons with a high GC content and of low occurrence in E. coli were eliminated. We incorporated a stem-loop circuit, devised to negatively control the expression of this highly toxic gene by partially hiding the ribosome-binding site (RBS) and START codon in mRNA secondary structures. Despite having optimized 59% of codons, the amount of produced RM.TthHB27I protein was similar for both recombinant tthHB27IRM gene variants. Moreover, the recombinant wt RM.TthHB27I is very unstable, while the RM.TthHB27I resulting from the expression of the synthetic gene exhibited enzymatic activities and stability equal to the native thermozyme isolated from T. thermophilus. Thus, we have developed an efficient purification protocol using the synthetic tthHB27IRM gene variant only. This suggests the effect of co-translational folding kinetics, possibly affected by the frequency of translational errors. The availability of active RM.TthHB27I is of practical importance in molecular biotechnology, extending the palette of available REase specificities.
获得耐热酶（热酶）是生物技术的一个重要方面。由于嗜热生物已经使它们的基因组适应了高温，因此在中温生物中克隆它们的基因表达是有问题的。这主要是由于它们的高 GC 含量，导致形成不利于大肠杆菌（E. coli）中 mRNA 结构和密码子使用的二级结构。RM.TthHB27I 是一种多功能热酶家族的成员，包含在单个多肽中的限制内切酶（REase）和甲基转移酶（MTase）。Thermus thermophilus HB27（T. thermophilus）产生具有独特 DNA 切割特异性的低量 RM.TthHB27I。我们之前已经将野生型（wt）基因克隆到大肠杆菌中，这使 RM.TthHB27I 的产量增加了 100 多倍。然而，对于在 T7 启动子下表达的 ORF，其酶活性极低。我们使用修改后的“密码子随机化”策略设计并克隆了完全合成的 tthHB27IRM 基因。消除了 GC 含量高且在大肠杆菌中出现频率低的密码子。我们设计了一个茎环回路，通过部分隐藏核糖体结合位点（RBS）和 mRNA 二级结构中的起始密码子，来负调控这个高度毒性基因的表达。尽管优化了 59%的密码子，但两种重组 tthHB27IRM 基因变体产生的 RM.TthHB27I 蛋白的量相似。此外，重组 wt RM.TthHB27I 非常不稳定，而表达合成基因产生的 RM.TthHB27I 表现出与从 T. thermophilus 分离的天然热酶相当的酶活性和稳定性。因此，我们仅使用合成的 tthHB27IRM 基因变体开发了一种有效的纯化方案。这表明共翻译折叠动力学的影响，可能受翻译错误频率的影响。活性 RM.TthHB27I 的可用性在分子生物技术中具有实际意义，扩展了可用的 REase 特异性的范围。
