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


RESULTS
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.


CONCLUSIONS
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.
Hillebrecht
Jason R
JR
New England Biolabs, 240 County Road, Ipswich, MA 01938, USA. jrhilleb@gmail.com
Chong
Shaorong
S
D000818:Animals; D002457:Cell Extracts; D002474:Cell-Free System; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000096:biosynthesis*; D005057:Eukaryotic Cells; D011387:Prokaryotic Cells; D014176:Protein Biosynthesis; D017510:Protein Folding; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis*; D012156:Reticulocytes / Q000378:metabolism*; D013997:Time Factors
D002457:Cell Extracts; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins