کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
9603161 | 43305 | 2005 | 6 صفحه PDF | دانلود رایگان |
عنوان انگلیسی مقاله ISI
In vivo directed evolution for thermostabilization of Escherichia coli hygromycin B phosphotransferase and the use of the gene as a selection marker in the host-vector system of Thermus thermophilus
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کلمات کلیدی
موضوعات مرتبط
مهندسی و علوم پایه
مهندسی شیمی
بیو مهندسی (مهندسی زیستی)
پیش نمایش صفحه اول مقاله

چکیده انگلیسی
An in vivo-directed evolutionary strategy was used to obtain a thermostabilized Escherichia coli hygromycin B phosphotransferase, using a host-vector system of Thermus thermophilus. Introduction of the mutant gene containing two amino acid substitutions, S52T and W238C, which was previously reported by Cannio et al. [J. Bacteriol., 180, 3237-3240 (1998)], did not confer hygromycin resistance on T. thermophilus cells at 55°C; however, five spontaneously-generated independent mutants were obtained by selection of the transformants at this temperature. Each mutant gene contained one amino acid substitution of either A118V or T246A. Further selection with increasing temperature, at 58°C and then 61°C, led to acquisition of three more substitutions: D20G, S225P and Q226L. These mutations cumulatively influenced the maximum growth temperature of the T. thermophilus transformants in the presence of hygromycin; T. thermophilus carrying a mutant gene containing all the five substitutions was able to grow at up to 67°C. This mutant gene, hph5, proved useful as a selection marker in the T. thermophilus host-vector system, either on the plasmid or by genome integration, at temperatures up to 65°C.
ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Journal of Bioscience and Bioengineering - Volume 100, Issue 2, August 2005, Pages 158-163
Journal: Journal of Bioscience and Bioengineering - Volume 100, Issue 2, August 2005, Pages 158-163
نویسندگان
Akira Nakamura, Yasuaki Takakura, Hideo Kobayashi, Takayuki Hoshino,