High-yield Escherichia coli -based cell-free expression of human proteins
Michel, Erich ; Wüthrich, Kurt
In: Journal of Biomolecular NMR, 2012, vol. 53, no. 1, p. 43-51
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- Production of sufficient amounts of human proteins is a frequent bottleneck in structural biology. Here we describe an Escherichia coli-based cell-free system which yields mg-quantities of human proteins in N-terminal fusion constructs with the GB1 domain, which show significantly increased translation efficiency. A newly generated E. coli BL21 (DE3) RIPL-Star strain was used, which contains a variant RNase E with reduced activity and an excess of rare-codon tRNAs, and is devoid of lon and ompT protease activity. In the implementation of the expression system we used freshly in-house prepared cell extract. Batch-mode cell-free expression with this setup was up to twofold more economical than continuous-exchange expression, with yields of 0.2-0.9mg of purified protein per mL of reaction mixture. Native folding of the proteins thus obtained is documented with 2D [15N,1H]-HSQC NMR