Widening the bottleneck: increasing success in protein expression and purification.
The number of variables at play in the expression and purification of a single protein dwarf those involved in sequencing a genome. Although certain trends are apparent, there is no one-size-fits-all approach to the process of purifying proteins. Thus, whereas numerous genome sequencing projects are providing an overwhelming number of interesting open reading frames for structural biologists to study, fully realizing the potential of this resource is still only a distant hope. We will discuss several current approaches to high throughput expression and purification as well as strategies that have served us well to quickly identify lead protein expression constructs in the context of a core service protein expression and purification laboratory. The use of the baculovirus expression vector system and implementation of a purification screening method will be emphasized.
Hopkins
Ralph
R
Protein Expression Laboratory, SAIC-Frederick, Inc., NCI-Frederick, Frederick, MD 21702, USA.
Esposito
Dominic
D
Gillette
William
W
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D040901:Proteomics / Q000379:methods; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015203:Reproducibility of Results; D018411:Spodoptera
D011506:Proteins; D011994:Recombinant Proteins