title: A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins.
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.
authors: J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA.
authors: Keehwan
authors: K
authors: Kwon
authors: Rembert
authors: R
authors: Pieper
authors: Shamira
authors: S
authors: Shallom
authors: Carissa
authors: C
authors: Grose
authors: Erika
authors: E
authors: Kwon
authors: Yu
authors: Y
authors: Do
authors: Saeeda
authors: S
authors: Latham
authors: Hamid
authors: H
authors: Alami
authors: Shih-Ting
authors: ST
authors: Huang
authors: Christine
authors: C
authors: Gatlin
authors: Leka
authors: L
authors: Papazisi
authors: Robert
authors: R
authors: Fleischmann
authors: Scott
authors: S
authors: Peterson
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003062:Codon; D016680:Genome, Bacterial; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D003062:Codon; D011994:Recombinant Proteins