Structural genomics efforts at the Chinese Academy of Sciences and Peking University

Structural genomics efforts at the Chinese Academy of Sciences and Peking University are reported in this article. The major targets for the structural genomics project are targeted proteins expressed in human hematopoietic stem/progenitor cells, proteins related to blood diseases and other human proteins. Up to now 328 target genes have been constructed in expression vectors. Among them, more than 50% genes have been expressed in Escherichia coli, approximately 25% of the resulting proteins are soluble, and 35 proteins have been purified. Crystallization, data collection and structure determination are continuing. Experiences accumulated during this initial stage are useful for designing and applying high-throughput approaches in structural genomics.Abbreviations: NSFC, National Natural Science foundation of China; MOST, Ministry of Science and Technology of China; CAS, Chinese Academy of Sciences; NSRL, National Synchrotron Radiation Laboratory in Hefei; BSRF, Beijing Synchrotron Radiation Facilities; HSPC, Hematopoietic stem/progenitor cells; APL, acute promyelocytic leukemia; ATRA, all-trans retinoic acid; COG, Cluster of Orthologous Groups of proteins.     

High‐throughput process development of purification alternatives for the protein avidin

With an increased number of applications in the field of the avidin‐biotin technology, the resulting demand for highly‐purified protein avidin has drawn our attention to the purification process of avidin that naturally occurs in chicken egg white. The high‐throughput process development (HTPD) methodology was exploited, in order to evaluate purification process alternatives to commonly used ion‐exchange chromatography. In a high‐throughput format, process parameters for aqueous two‐phase extraction, selective precipitation with salts and polyethylene glycol, and hydrophobic interaction and mixed‐mode column chromatography experiments were performed. The HTPD strategy was complemented by a high‐throughput tandem high‐performance liquid chromatography assay for protein quantification. Suitable conditions for the separation of avidin from the major impurities ovalbumin, ovomucoid, ovotransferrin, and lysozyme were identified in the screening experiments. By combination of polyethylene glycol precipitation with subsequent resolubilization and separation in a polyethylene glycol/sulfate/sodium chloride two‐phase system an avidin purity of 77% was obtained with a yield >90% while at the same time achieving a significant reduction of the process volume. The two‐phase extraction and precipitation results were largely confirmed in larger scale with scale‐up factors of 230 and 133, respectively. Seamless processing of the avidin enriched bottom phase was found feasible by using mixed‐mode chromatography. By gradient elution a final avidin purity of at least 97% and yield >90% was obtained in the elution pool. The presented identification of a new and beneficial alternative for the purification of the high value protein thus represents a successful implementation of HTPD for an industrially relevant purification task. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:957–973, 2015     

On the structure of hisH: protein structure prediction in the context of structural and functional genomics

We predict a structure of the glutamine amidotransferase subunit (hisH) of imidazole glycerol phosphate synthase (IGPS) which catalyzes the fifth step of the histidine biosynthesis in Escherichia coli. The model is constructed using an energy-based threading program augmented by a multiple sequence to structure profile analysis. In developing our model we identified a conserved core region within hisH and a variable domain which is the likely site of interaction with the synthase subunit (hisF) of IGPS. Information available from structural and functional genomics studies was used to improve the structure prediction, to discuss parallels between histidine biosynthesis and other amino acid and nucleotide metabolic pathways, and to better understand the protein-protein interactions between the hisH and hisF domains of IGPS. This work allows us to develop a preliminary model for the structure of the entire IGPS holoenzyme.