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 H-1/C-13 methyl protonated proteins from 100 ml cultures in M9 ++ /D2O medium induced at high (OD600 similar to 10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of H-2/N-15/C-13 and H-1/C-13 labeled proteins, yields comparable quantities of protein from 100 ml cell culture to those obtained using a conventional 1 L culture with M9/D2O medium, while using three-fold less alpha-ketoisovaleric (1,2,3,4-C-13(4); 3,4 ',4 ',4 '-d(4)) and alpha-ketobutyric (C-13(4); 3,3-d(2)) acid precursors.

Automated summary

The presented protocol allows for the expression of perdeuterated, Ile/Leu/Val H-1/C-13 methyl protonated proteins from 100 ml cultures in a cost-effective manner with comparable protein yields to conventional 1 L cultures, while using three-fold less precursors.