Structural genomics studies of human caries pathogen Streptococcus mutans.
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.
State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing, 100871, China, lilf@pku.edu.cn.
Lanfen
L
Li
Jie
J
Nan
Dan
D
Li
Erik
E
Brostromer
Zixi
Z
Wang
Cong
C
Liu
Qiaoming
Q
Hou
Xuexin
X
Fan
Zhaoyang
Z
Ye
Xiao-Dong
XD
Su
D001426:Bacterial Proteins / Q000235:genetics / Q000648:ultrastructure*; D003001:Cloning, Molecular; D019295:Computational Biology; D003460:Crystallization / Q000379:methods; D018360:Crystallography, X-Ray; D003731:Dental Caries / Q000382:microbiology*; D016680:Genome, Bacterial / Q000235:genetics; D023281:Genomics / Q000379:methods; D006801:Humans; D007090:Image Interpretation, Computer-Assisted; D040901:Proteomics / Q000379:methods; D013295:Streptococcus mutans / Q000235:genetics*
D001426:Bacterial Proteins