Editing S30 E. coli extract (section)

== PROTOCOL 5 ==

For small-scale in vitro synthesis of unlabeled proteins, the inner-chamber reaction mixtures (total volume 100 ll) contained 55 mM HEPES/ KOH (pH 7.5), 1.7 mM dithiothreitol, 1.2 mM ATP, 0.8 mM each of CTP, GTP and UTP, 0.64 mM 3¢,5¢-cyclic AMP, 68 lM folinic acid, 27.5 mM ammonium acetate, 208 mM potassium glutamate, 80 mM creatine phosphate, 25 lg creatine kinase, 1 mM of each of the twenty L-amino acids, 15 mM magnesium acetate, 17.5 lg E. coli total tRNA, 18 ll of Rosetta (kDE3)/pRARE S30K extract, 1.6 lg of supercoiled plasmid DNA, 150 units of RNase inhibitor, and either 9.3 lg of purified T7 RNAP or 3.2 lg of supercoiled plasmid pKO1166. Since T7 RNAP contains 100 alanine residues, the concentration of L-Ala was doubled when using pKO1166. Cell-free protein synthesis was carried out in Spectrapor 2 dialysis tubing with a nominal size cutoff of 12–14 kDa for 8 h at 37 C with gentle shaking in 3 ml of outer chamber solution. The inner chamber assembly and the outer chamber buffer were housed within a screw-capped 10 ml polypropylene tube. The outer chamber solution had the same composition as the inner chamber reaction mixture, except that S30K extract, tRNA, plasmid DNA, pKO1166 or T7 RNAP, creatine kinase and RNase inhibitor were omitted, and the concentration of magnesium acetate was increased to 19.3 mM. After incubation, the soluble and insoluble components of the inner chamber were separated by centrifugation (30,000 · g, 1 h at 4 C)