A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins |
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Authors: | Kwon Keehwan Pieper Rembert Shallom Shamira Grose Carissa Kwon Erika Do Yu Latham Saeeda Alami Hamid Huang Shih-Ting Gatlin Christine Papazisi Leka Fleischmann Robert Peterson Scott |
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Institution: | J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA. |
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Abstract: | We have developed and evaluated a highly parallel protein expression and purification system using ORFs derived from the pathogenic bacterium Streptococcus pneumoniae as a representative test case in conjunction with the Gateway cloning technology. Establishing high throughput protein production capability is essential for genome-wide characterization of protein function. In this study, we focused on protein expression and purification outcomes generated from an expression vector which encodes an NH(2)-terminal hexa-histidine tag and a COOH-terminal S-tag. Purified recombinant proteins were validated by SDS-PAGE, followed by in-gel digestion and identification by MALDI-TOF/TOF analysis. Starting with 1360 sequence-validated destination clones we examined correlation analyses of expression and solubility of a wide variety of recombinant proteins. In total, 428 purified proteins (31%) were recovered in soluble form. We describe a semi-quantitative scoring method using an S-tag assay to improve the throughput and efficiency of expression and solubility studies for recombinant proteins. Given a relatively large dataset derived from proteins representing all functional groups in a microbial genome we correlated various protein characteristics as they relate to protein expression outcomes. |
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Keywords: | High throughput screening Protein expression Purification Genome-wide expression Codon usage His-tag S-tag assay Correlation analysis |
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