Prolonged production of proteins in a cell-free protein synthesis system using polymeric carbohydrates as an energy source

Cell-free protein synthesis: Applications come of age

“…Thus, extract preparation is simple and inexpensive. Second, E. coli based systems generally achieve the highest protein yields, from hundreds of micrograms per milliliter to milligrams per milliliter in a batch reaction, depending on the protein of interest ( e.g ., 1.7 mg mL -1 chloramphenicol acetyl transferase (Kim et al , 2011), 0.7 mg mL -1 human granulocyte-macrophage colony-stimulating factor (Zawada et al , 2011), and 0.022 mg mL -1 FeFe hydrogenase (Boyer et al , 2008)). Third, the reaction cost of the E. coli system is the lowest.…”

“…Substituting nucleoside monophosphates (NMPs) for nucleoside triphosphates (NTPs) further reduced energy costs while maintaining high protein yields (Calhoun and Swartz, 2005a, Jewett et al , 2008). Complementary efforts have more recently utilized polymeric carbohydrates such as maltodextran (Wang and Zhang, 2009) and soluble starch or glycogen (Kim et al , 2011) as energy substrates because they are slowly metabolized. A key advantage of these energy substrates is that environmental factors like pH and inorganic phosphate concentration are more stable, which can lead to higher protein expression.…”

A cost-effective polyphosphate-based metabolism fuels an all E. coli cell-free expression system

“…Cytoplasmic enzymes, present in the cellular extract, are exploited to regenerate nucleosides triphosphate, such as ATP and GTP (Swartz, 2012). Another significant reduction of the reaction cost is obtained by exploiting the central metabolism using polysaccharides, glucose, pyruvate, (Calhoun and Swartz, 2005;Jewett and Swartz, 2004b;Kim et al, 2011;Wang and Zhang, 2009), and oxidative phosphorylation with glutamate (Jewett et al, 2008). These novel systems bypass the Contents lists available at ScienceDirect journal homepage: www.elsevier.com/locate/ymben substrate level-phosphorylation based on expensive phosphate donors energy sources and externally added kinases (Kim and Swartz, 2001;Kim et al, 2006;Sitaraman et al, 2004).…”

Emerging technology of in situ cell free expression protein microarrays

“…More recently, higher protein levels have been achieved by supplying extraenergy for protein biosynthesis in cell free expression system (Kim et al, 2011). Other attempts deal with scaling up the cell extract production (Zawada et al, 2011), using diverse fusion tags (Kralicek et al, 2011), a cell mimic device (Siuti et al, 2011) or using microporus microfluidic devices that will give a cleaner and high density protein microarray.…”