Structural genomics studies of human caries pathogen Streptococcus mutans

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.

Codon usage clusters correlation: towards protein solubility prediction in heterologous expression systems in E. coli

“…We show for the first time a quantitative study of the total expression and solubility of thirty proteins from a psychrotolerant organism. We found that 50% of the expressed proteins could be classified as soluble, being this value remarkably similar to that previously described for proteins of thermophilic and mesophilic organisms 11 , 12 . In addition, we evaluated the influence of different factors, known to modulate heterologous protein production, on the experimental expression and solubility of our dataset.…”

Protein crystallography from the perspective of technology developments

“…The systematic use of selenomethionine as an anomalously scatterer that could be readily incorporated into proteins as a powerful agent for obtaining crystallographic phases has dramatically changed structural biology and greatly contributed to the vision and realization of Structural Genomics [72]. The development of algorithms for automatic interpretation of macromolecular diffraction data and generation of atomic, combined with the extensive automation in gene sequencing and the beginnings of automation in gene cloning and protein expression suggested to many that ever more automated and rapid structure determination could be carried out [182, 183, 184, 185, 186]. …”

“…We have applied the SG approaches to the structural and functional characterization of proteins from a dental caries pathogen Streptococcus mutans . Using the platform, we have cloned about 1,400 genes encoding non-membrane proteins from S. mutans genome containing 1963 ORFs [183, 203] into a variety of pET vectors using mainly conventional cloning techniques. We then over-expressed and purified soluble proteins by using an E. coli expression system.…”