Publications
Citation
Kwon, K., Pieper, R., Shallom, S., Grose, C., Kwon, E., Do, Y., Latham, S., Alami, H., Huang, S. T., Gatlin, C., Papazisi, L., Fleischmann, R., Peterson, S.
A Correlation Analysis of Protein Characteristics Associated With Genome-wide High Throughput Expression and Solubility of Streptococcus pneumoniae Proteins
Protein Expr Purif. 2007 Oct 01; 55(2): 368-78.
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.