• Journal of Microbiology and Biotechnology
• /
• 제16권9호
• /
• pp.1362-1368
• /
• 2006

A new constitutive episomal expression vector, pGAPZ-E, was constructed and used for initial screening of eukaryotic target gene expression in Pichia pastoris. Two reporter genes such as beta-galactosidase gene and GFPuv gene were overexpressed in P. pastoris. The expression level of the episomal pGAPZ-E strain was higher than that of the integrated form when the beta-galactosidase gene was used as the reporter gene in P. pastoris X33. The avoiding of both the integration procedure and an induction step simplified the overall screening process for eukaryotic target gene expression in P. pastoris. Nine human protein targets from the Core 50, family of Northeast Structural Genomics Consortium (http://www.nesg.org), which were intractable when expressed in E. coli, were subjected to rapid screening for soluble expression in P. pastoris. HR547, HR919, and HR1697 human proteins, which had previously been found to express poorly or to be insoluble in E. coli, expressed in soluble form in P. pastoris. Therefore, the new episomal GAP promoter vector provides a convenient and alternative system for high-throughput screening of eukaryotic protein expression in P. pastoris.

• BMB Reports
• /
• 제43권5호
• /
• pp.319-324
• /
• 2010

Experimental bioinformatics data obtained from an E. coli cell-based eukaryotic protein purification experiment were analyzed in order to identify any bottleneck as well as the factors affecting the target purification. All targets were expressed as His-tagged maltose-binding protein (MBP) fusion constructs and were initially purified by immobilized metal affinity chromatography (IMAC). The targets were subsequently separated from the His-tagged MBP through TEV protease cleavage followed by a second IMAC isolation. Of the 743 total purification trials, 342 yielded more than 3 mg of target proteins for structural studies. The major reason for failure of target purification was poor TEV proteolysis. The overall success rate for target purification decreased linearly as cysteine content or isoelectric point (pI) of the target increased. This pattern of pI versus overall success rate strongly suggests that pI should be incorporated into target scoring criteria with a threshold value.

• Interdisciplinary Bio Central
• /
• 제3권4호
• /
• pp.14.1-14.9
• /
• 2011

Protein homology detection is an important issue in comparative genomics. Because of the exponential growth of sequence databases, fast and efficient homology detection tools are urgently needed. Currently, for homology detection, sequence comparison methods using local alignment such as BLAST are generally used as they give a reasonable measure for sequence similarity. However, these methods have drawbacks in offering overall sequence similarity, especially in dealing with eukaryotic genomes that often contain many insertions and duplications on sequences. Also these methods do not provide the explicit models for speciation, thus it is difficult to interpret their similarity measure into homology detection. Here, we present a novel method based on Word Conservation Score (WCS) to address the current limitations of homology detection. Instead of counting each amino acid, we adopted the concept of 'Word' to compare sequences. WCS measures overall sequence similarity by comparing word contents, which is much faster than BLAST comparisons. Furthermore, evolutionary distance between homologous sequences could be measured by WCS. Therefore, we expect that sequence comparison with WCS is useful for the multiple-species-comparisons of large genomes. In the performance comparisons on protein structural classifications, our method showed a considerable improvement over BLAST. Our method found bigger micro-syntenic blocks which consist of orthologs with conserved gene order. By testing on various datasets, we showed that WCS gives faster and better overall similarity measure compared to BLAST.