Abstract
Gram-positive bacterium Streptococcus mutans is the primary causative agent of human dental caries. To better understand this pathogen at the atomic structure level and to establish potential drug and vaccine targets, we have carried out structural genomics research since 2005. To achieve the goal, we have developed various in-house automation systems including novel high-throughput crystallization equipment and methods, based on which a large-scale, high-efficiency and low-cost platform has been establish in our laboratory. From a total of 1,963 annotated open reading frames, 1,391 non-membrane targets were selected prioritized by protein sequence similarities to unknown structures, and clustered by restriction sites to allow for cost-effective high-throughput conventional cloning. Selected proteins were over-expressed in different strains of Escherichia coli. Clones expressed soluble proteins were selected, expanded, and expressed proteins were purified and subjected to crystallization trials. Finally, protein crystals were subjected to X-ray analysis and structures were determined by crystallographic methods. Using the previously established procedures, we have so far obtained more than 200 kinds of protein crystals and 100 kinds of crystal structures involved in different biological pathways. In this paper we demonstrate and review a possibility of performing structural genomics studies at moderate laboratory scale. Furthermore, the techniques and methods developed in our study can be widely applied to conventional structural biology research practice.
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Acknowledgments
This work has been supported by the National Natural Science Foundation of China (Grant No. 30530190), and 985 and 211 grants from Peking University. Due to space limit, it is difficult to list all people that have been involved in the project. Hereby, we would like to acknowledge all the group members that have contributed to this project during the period of 2005-2010, particularly Drs. Yuhe Liang, Xiaoyan Zhang, Na Yang, Yamei Yu, Wei Mi, Yanfeng Zhou, Jian Lei, Juan Wang, Xiaoyan Liu, Guilan Li, Kaituo Wang, Linglong Ma, Xiaojun Wang, Tianmin Fu, Xiang Liu, Xiangyu Liu, Mingjing Feng, Yuehua Liu, Xiaofang Cao, Dong Xing, Yihe huang, Yanli Cao, Yuhui Dong, Zengqiang Gao, Haifeng hou, and Shicheng Wei.
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Li, L., Nan, J., Li, D. et al. Structural genomics studies of human caries pathogen Streptococcus mutans . J Struct Funct Genomics 15, 91–99 (2014). https://doi.org/10.1007/s10969-014-9172-3
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DOI: https://doi.org/10.1007/s10969-014-9172-3