Abstract
Recombinant expression of proteins of interest in Escherichia coli is an important tool in the determination of protein structure. However, lack of expression and insolubility remain significant challenges to the expression and crystallization of these proteins. The SSGCID program uses a wheat germ cell-free expression system as a rescue pathway for proteins that are either not expressed or insoluble when produced in E. coli. Testing indicates that the system is a valuable tool for these protein targets. Further increases in solubility were obtained by the addition of the NVoy polymer reagent to the reaction mixture. These data indicate that this eukaryotic cell-free expression system has a high success rate and that the addition of specific reagents can increase the yield of soluble protein.
Highlights
20% of the protein targets were soluble when expressed in E. coli, 52% were insoluble and the remaining 22% were not expressed (Table 1)
Detectable protein was achieved for 87% of targets based on Western blot analysis (Tables 1 and 2). 83% of the targets expressing insoluble protein in E. coli expressed soluble protein in the cell-free system (Table 1)
1030 Guild et al Wheat germ cell-free expression system. This analysis of expression of proteins from the Seattle Structural Genomics Center for Infectious Disease (SSGCID) pipeline in a eukaryotic in vitro system validates the use of the wheat germ cellfree system for expression of proteins that are either not expressed or are primarily insoluble when expressed in E. coli
Summary
The wheat germ cell-free expression system combines the advantages of cell-free and eukaryotic systems and is well suited for expression of difficult-to-express proteins such as disulfide-bond-containing or integral membrane proteins (Endo & Sawasaki, 2006; Kawasaki et al, 2003; Spirin, 2004; Vinarov, Loushin Newman & Markley, 2006; Vinarov, Loushin Newman, Tyler et al, 2006; Klammt et al, 2006; Tyler et al, 2005) This system has been used as a rescue pathway for human proteins that are not soluble in both in vivo and in vitro E. coli systems (Langlais et al, 2007)
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