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

J Biomol NMR. 2021 Mar;75(2-3):83-87. doi: 10.1007/s10858-021-00357-x. Epub 2021 Feb 4.

Abstract

A simple and cost-effective protocol is presented for expression of perdeuterated, Ile/Leu/Val 1H/13C methyl protonated proteins from 100 ml cultures in M9 ++ /D2O medium induced at high (OD600 ~ 10) cell density in shaker flasks. This protocol, which is an extension of our previous protocols for expression of 2H/15N/13C and 1H/13C 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 α-ketoisovaleric (1,2,3,4-13C4; 3,4',4',4'-d4) and α-ketobutyric (13C4; 3,3-d2) acid precursors.

Keywords: High cell-density induction; M9 ++ medium; NMR; Perdeuterated and methyl protonated proteins; Protein expression; Shaker flask.