Comparison of small- and large-scale expression of selected Pyrococcus furiosus genes as an aid to high-throughput protein production.
As the natural extension of the genomic sequencing projects, the goal of the various world-wide Structural Genomics projects is development of techniques for high throughput (HTP) cloning, protein overexpression, purification and structural determination, with the ultimate goal of determining all possible protein structures. Rapid (small-scale) screening of potential expression clones under different growth conditions is presumed to be possible and a viable way to increase throughput of protein expression. In order to test the utility of screening for soluble, heterologous protein expression, we have compared the production of recombinant proteins on a small scale (1 ml cultures in 96-well plates) in Escherichia coli under two growth conditions [a rich medium and a defined (minimal) medium] using an enzyme-linked immunosorbent assay (ELISA) against the affinity tag, with the amount of recombinant protein produced during the large-scale (500 ml) growth of E. coli. The large-scale expression products were examined after a single step affinity purification by visualization on SDS-PAGE gels. Of the open reading frames that were successfully expressed on the 1 ml scale as judged by immunodetection, 80% of them successfully scaled-up to 500 ml in a rich medium and 81% of them scaled-up in a defined medium. This is significantly higher than would be expected by a randomly selected expression condition and validates the use of small-scale expression as a screening tool for more efficient protein production.
Sugar
Frank J
FJ
Department of Biochemistry and Molecular Biology, Southeastern Collaboratory for Structural Genomics, University of Georgia, Athens, GA 30602, USA.
Jenney
Francis E
FE
Poole
Farris L
FL
Brereton
Phillip S
PS
Izumi
Michi
M
Shah
Claudia
C
Adams
Michael W W
MW
D001426:Bacterial Proteins / Q000302:isolation & purification* / Q000378:metabolism; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli; D015870:Gene Expression; D016366:Open Reading Frames / Q000235:genetics; D040901:Proteomics / Q000379:methods*; D020027:Pyrococcus furiosus / Q000235:genetics*; D011994:Recombinant Proteins / Q000302:isolation & purification* / Q000378:metabolism
D001426:Bacterial Proteins; D003470:Culture Media; D011994:Recombinant Proteins