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Quantitative polysome analysis identifies limitations in bacterial cell‐free protein synthesis
Author(s) -
Underwood Kelly A.,
Swartz James R.,
Puglisi Joseph D.
Publication year - 2005
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20529
Subject(s) - polysome , protein biosynthesis , elongation , ribosome , translation (biology) , eukaryotic translation , ef tu , cell free protein synthesis , biochemistry , biophysics , messenger rna , elongation factor , initiation factor , chemistry , biology , microbiology and biotechnology , rna , materials science , gene , ultimate tensile strength , metallurgy
Cell‐free protein synthesis (CFPS) is becoming increasingly used for protein production as yields increase and costs decrease. CFPS optimization efforts have focused primarily on energy supply and small molecule metabolism, though little is known about the protein synthesis machinery or what limits protein synthesis rates. Here, quantitative polysome profile analysis was used to characterize cell‐free translation, thereby elucidating many kinetic parameters. The ribosome concentration in Escherichia coli ‐based CFPS reactions was 1.6 ± 0.1 μM, with 72 ± 4% actively translating at maximal protein synthesis rate. A translation elongation rate of 1.5 ± 0.2 amino acids per second per ribosome and an initiation rate of 8.2 × 10 −9 ± 0.3 × 10 −9 M/s, which correlates to, on average, one initiation every 60 ± 9 s per mRNA, were determined. The measured CFPS initiation and elongation rates are an order of magnitude lower than the in vivo rates and further analysis identified elongation as the major limitation. Adding purified elongation factors (EFs) to CFPS reactions increased the ribosome elongation rate and protein synthesis rates and yields, as well as the translation initiation rate, indicating a possible coupling between initiation and elongation. Further examination of translation initiation in the cell‐free system showed that the first initiation on an mRNA is slower than subsequent initiations. Our results demonstrate that polysome analysis is a valid tool to characterize cell‐free translation and to identify limiting steps, that dilution of translation factors is a limitation of CFPS, and that CFPS is a useful platform for making novel observations about translation. © 2005 Wiley Periodicals, Inc.