Selective and efficient extraction of recombinant proteins from the periplasm of Escherichia coli using low concentrations of chemicals

Experiments were conducted to determine chemicals at low concentrations, which can be utilized for selective release of periplasmic proteins. It was revealed that 80-100 % of the activity of alpha-amylase, beta-lactamase, and Fab D1.3 was retained in the presence of 0.05 and 0.1 % Triton X-100, 0.1 % Tween 20, 0.1 % DOC, 0.01 % BAC, 0.01 % CTAB, 10 mM EDTA, 1 mM and 10 mM DEA, 10 mM NTA, 0.1 and 1 % SHMP, 200 mM urea, 100-500 mM GndCl, and 1 % solvents (hexane, xylene, toluene, benzene, pyridine and isoamyl alcohol). Performance of these chemicals, recognized as generally safe, for selective release of proteins from the periplasm of Escherichia coli was investigated. DOC was a general and very efficient agent, and at concentrations as low as 0.05, 0.1, and 0.025 %, released beta-lactamase, alpha-amylase, and Fab D1.3 selectively with yield factors of 2.7, 2.3, and 3.6 times greater than osmotic shock procedure, respectively. EDTA (1 and 10 mM) discharged Fab D1.3 with efficiency more than osmotic shock (target protein yield of 110 and 138 %, correspondingly). Isoamyl alcohol (10 % v/v) was effective for periplasmic release of alpha-amylase and particularly Fab D1.3, with target protein yields of 75 and 168 %, respectively.