A correlation analysis of protein characteristics associated with genome-wide high throughput expression and solubility of Streptococcus pneumoniae proteins.
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.
Kwon
Keehwan
K
J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA.
Pieper
Rembert
R
Shallom
Shamira
S
Grose
Carissa
C
Kwon
Erika
E
Do
Yu
Y
Latham
Saeeda
S
Alami
Hamid
H
Huang
Shih-Ting
ST
Gatlin
Christine
C
Papazisi
Leka
L
Fleischmann
Robert
R
Peterson
Scott
S
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*
D001426:Bacterial Proteins; D003062:Codon; D011994:Recombinant Proteins