A simple and cost-effective protocol for high-yield expression of deuterated and selectively isoleucine/leucine/valine methyl protonated proteins in Escherichia coli grown in shaker flasks

A simple and cost-effective protocol is presented for expression of perdeuterated, Ile/Leu/Val (1)H/(13)C methyl protonated proteins from 100 ml cultures in M9 ++ /D(2)O medium induced at high (OD(600) ~ 10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of (2)H/(15)N/(13)C and (1)H/(13)C labeled proteins, yields comparable quantities of protein from 100 ml cell culture to those obtained using a conventional 1 L culture with M9/D(2)O medium, while using three-fold less α-ketoisovaleric (1,2,3,4-(13)C(4); 3,4′,4′,4′-d(4)) and α-ketobutyric ((13)C(4); 3,3-d(2)) acid precursors.