A comparative study of protein synthesis in in vitro systems: from the prokaryotic reconstituted to the eukaryotic extract-based

New England Biolabs, 240 County Road, Ipswich, MA 01938, USA.
BMC Biotechnology (Impact Factor: 2.03). 02/2008; 8(1):58. DOI: 10.1186/1472-6750-8-58
Source: PubMed


Cell-free protein synthesis is not only a rapid and high throughput technology to obtain proteins from their genes, but also provides an in vitro platform to study protein translation and folding. A detailed comparison of in vitro protein synthesis in different cell-free systems may provide insights to their biological differences and guidelines for their applications.
Protein synthesis was investigated in vitro in a reconstituted prokaryotic system, a S30 extract-based system and a eukaryotic system. Compared to the S30 system, protein synthesis in the reconstituted system resulted in a reduced yield, and was more cold-sensitive. Supplementing the reconstituted system with fractions from a size-exclusion separation of the S30 extract significantly increased the yield and activity, to a level close to that of the S30 system. Though protein synthesis in both prokaryotic and eukaryotic systems showed no significant differences for eukaryotic reporter proteins, drastic differences were observed when an artificial fusion protein was synthesized in vitro. The prokaryotic systems failed to synthesize and correctly fold a significant amount of the full-length fusion protein, even when supplemented with the eukaryotic lysate. The active full-length fusion protein was synthesized only in the eukaryotic system.
The reconstituted bacterial system is sufficient but not efficient in protein synthesis. The S30 system by comparison contains additional cellular factors capable of enhancing protein translation and folding. The eukaryotic translation machinery may have evolved from its prokaryotic counterpart in order to translate more complex (difficult-to-translate) templates into active proteins.

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    • "Table 1 systematically compares the PURE system with a commercialized E. coli crude extract CFPS (the 5 PRIME RTS system). The ribosome concentration is ∼2.4 µM in the PURE system and ∼1.6 µM in the crude extract system, The PURE system's productivity as measured by the yield of active firefly luciferase is 3 fold lower [19] while its cost is ∼4 times higher per gram of protein produced than those of the crude extract system. Additionally, the PURE system's preparation procedure, which involves multiple column based purifications, is more labor and time consuming than the preparation of crude extract system. "
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    PLoS ONE 09/2014; 9(9):e106232. DOI:10.1371/journal.pone.0106232 · 3.23 Impact Factor
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    • "Cell-free protein expression is a rapid and highthroughput technology used to express proteins from the genes of interest by involving cell-free lysate from an Escherichia coli cell, rabbit reticulocytes, or wheat germ as the protein translation machinery. Cell-free systems have been used extensively for studies on translational control and protein production (Laxminarayana et al., 2002; Szamecz et al., 2008) at mg per mL levels (Hillebrecht and Chong, 2008). It has several applications including proteomics, protein folding and amino acid functional studies. "
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    • "Commercially available cell-free systems are derived from Escherichia coli extracts, wheat germ extracts (WGE), rabbit reticulocytes (RRL), and lysates from insect cells (Spodoptera frugiperda cells Sf21, Qiagen EasyXpress Insect Kit II). Cell extracts from E. coli provide high protein yield [8], but a major drawback of these lysates is the absence of the glycosylation machinery. Nevertheless, it was shown that extracts from E. coli transformed with the entire protein glycosylation locus gene cluster from gram negative bacteria Campylobacter jejuni can perform N-linked protein glycosylation [9] [10]. "
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    ABSTRACT: Cell-free protein synthesis (CFPS) is a valuable method for the fast expression of difficult-to-express proteins as well as posttranslationally modified proteins. Since cell-free systems circumvent possible cytotoxic effects caused by protein overexpres- sion in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and func- tionally diverse proteins. Our eukaryotic cell-free translation systems are capable to provide high molecular weight membrane proteins, fluorescent-labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis.
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