Reduction of protein degradation by use of protease-deficient mutants in cell-free protein synthesis system of Escherichia coli.
In an Escherichia coli in vitro transcription/translation system, the degradation of produced proteins is often caused by endogenous proteases from E. coli extracts. To reduce the extent of this degradation, several extracts were prepared from E. coli mutants that genetically lacked DegP, OmpT, or Lon proteases. Then, these extracts were used with 14C-leucine in a system for synthesizing single-chain Fv against gp120 (anti-gp120), and phospholipase D (PLD) of Streptomyces antibioticus. The proteins synthesized in vitro were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and autoradiography. The use of extracts from mutants that were deficient in the structural genes encoding OmpT and Lon markedly repressed the degradation of anti-gp120. Similarly, extracts from degP- and ompT-deleted mutants were able to significantly stabilize in vitro-synthesized PLD, which otherwise disappeared within 30 min. Such protease-deficient mutants were suggested to be useful for preventing the degradation of heterologous proteins in in vitro systems.
Jiang
Xiuping
X
Laboratory of Molecular Biotechnology, Graduate School of Biological and Agricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan.
Oohira
Kazuyo
K
Iwasaki
Yugo
Y
Nakano
Hideo
H
Ichihara
Shigeyuki
S
Yamane
Tsuneo
T