Cell‐free production and stable‐isotope labeling of milligram quantities of proteins

We have improved the productivity of an Escherichia coli cell‐free protein synthesis system. First, creatine phosphate and creatine kinase were used as the energy source regeneration system, and the other components of the reaction mixture were optimized. Second, the E. coli S30 cell extract was condensed by dialysis against a polyethylene glycol solution to increase the rate of synthesis. Third, during the protein synthesis, the reaction mixture was dialyzed against a low‐molecular‐weight substrate solution to prolong the reaction. Thus, the yield of chloramphenicol acetyltransferase was raised to 6 mg/ml of reaction mixture. Stable‐isotope labeling of a protein with 13C/15N‐labeled amino acids for NMR spectroscopy was achieved by this method.

[1]  H. Pelham,et al.  An efficient mRNA-dependent translation system from reticulocyte lysates. , 1976, European journal of biochemistry.

[2]  O. W. Odom,et al.  An effect of polyethylene glycol 8000 on protein mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a method for eliminating this effect. , 1997, Analytical biochemistry.

[3]  H. Khorana,et al.  Site-directed isotope labelling and FTIR spectroscopy of bacteriorhodopsin , 1994, Nature Structural Biology.

[4]  A. Spirin,et al.  Acetyl phosphate as an energy source for bacterial cell-free translation systems. , 1995, Analytical biochemistry.

[5]  T. Yamane,et al.  Highly productive cell-free protein synthesis system using condensed wheat-germ extract , 1996 .

[6]  A. Spirin,et al.  A continuous cell-free translation system capable of producing polypeptides in high yield. , 1988, Science.

[7]  E D Laue,et al.  Regional polysterism in the GTP-bound form of the human c-Ha-Ras protein. , 1997, Biochemistry.

[8]  S. Yokoyama,et al.  Guanine-nucleotide binding activity, interaction with GTPase-activating protein and solution conformation of the human c-Ha-Ras protein catalytic domain are retained upon deletion of C-terminal 18 amino acid residues , 1992, Journal of protein chemistry.

[9]  T. Yamane,et al.  An increased rate of cell-free protein synthesis by condensing wheat-germ extract with ultrafiltration membranes. , 1994, Bioscience, biotechnology, and biochemistry.

[10]  E D Laue,et al.  Dual amino acid-selective and site-directed stable-isotope labeling of the human c-Ha-Ras protein by cell-free synthesis , 1998, Journal of biomolecular NMR.

[11]  H. Gross,et al.  Rapid and simple purification of T7 RNA polymerase. , 1991, Nucleic acids research.

[12]  S. Yoon,et al.  A modified Coomassie blue staining of proteins in polyacrylamide gels with Bismark brown R. , 1996, Analytical biochemistry.

[13]  T. Yamane,et al.  A long-lived batch reaction system of cell-free protein synthesis. , 1995, Analytical biochemistry.

[14]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[15]  C. Choi,et al.  A Semicontinuous Prokaryotic Coupled Transcription/Translation System Using a Dialysis Membrane , 1996, Biotechnology progress (Print).

[16]  W. V. Shaw.  [57] Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria , 1975 .

[17]  S. Yokoyama,et al.  Conformation of guanosine 5'-diphosphate as bound to a human c-Ha-ras mutant protein: a nuclear Overhauser effect study. , 1989, Biochemistry.

[18]  T. Kigawa,et al.  Cell-free synthesis and amino acid-selective stable isotope labeling of proteins for NMR analysis , 1995, Journal of biomolecular NMR.

[19]  T. Kigawa,et al.  A highly efficient cell-free protein synthesis system from Escherichia coli. , 1996, European journal of biochemistry.

[20]  S. Yokoyama,et al.  Sequence-specific 1H and 15N resonance assignments and secondary structure of GDP-bound human c-Ha-Ras protein in solution , 1993, Journal of biomolecular NMR.

[21]  P G Schultz,et al.  Biosynthetic method for introducing unnatural amino acids site-specifically into proteins. , 1991, Methods in enzymology.