Rapid purification of highly active ribosomes from Escherichia coli

✍ Scribed by Pierre C. Jelenc

• Publisher: Elsevier Science
• Year: 1980
• Tongue: English
• Weight: 1000 KB
• Volume: 105
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(80)90472-8

✦ Synopsis

Highly active salt-washed ribosomes from Escherichia coli are isolated by gel filtration on Sephacryl S-200 in a buffer containing 1 M ammonium chloride and putrescine, spermidine, calcium, and magnesium ions. Up to several hundred grams of cells can be processed in less than 24 h. The ribosome solution made 30% (v/v) in methanol is best stored as a liquid at -20°C. An improved poly(U)-dependent peptide synthesis assay is described in which phenylalanine polymerization is greatly enhanced while the misincorporation of leucine is kept at in vivo levels.

' Abbreviations used: polymix buffer, 5 tTIM W+, 0.5 mM Ca*+, 8 mM putrescine, 1 mM spermidine, 5 mM phosphate, 5 mM NW, 95 mM K+, 1 mM dithioerythritol, pH 7.5; tricine, N-tris(hydroxymethyl)methyl glycine; buffer I, same as polymix but with tricine instead of phosphate; buffer II, buffer I made 1 M in NH&l; EF-Tu, elongation factor Tu; EF-G, elongation factor G.