Optimization of an Escherichia coli system for cell‐free synthesis of selectively ¹⁵N‐labelled proteins for rapid analysis by NMR spectroscopy

Abstract

Cell-free protein synthesis offers rapid access to proteins that are selectively labelled with [ 15 N]amino acids and suitable for analysis by NMR spectroscopy without chromatographic purification. A system based on an Escherichia coli cell extract was optimized with regard to protein yield and minimal usage of 15 N-labelled amino acid, and examined for the presence of metabolic by-products which could interfere with the NMR analysis. Yields of up to 1.8 mg of human cyclophilin A per mL of reaction medium were obtained by expression of a synthetic gene. Equivalent yields were obtained using transcription directed by either T7 or tandem phage k p R and p L promoters, when the reactions were supplemented with purified phage T7 or E. coli RNA polymerase. Nineteen samples, each selectively labelled with a different 15 N-enriched amino acid, were produced and analysed directly by NMR spectroscopy after ultracentrifugation. Cross-peaks from metabolic by-products were evident in the 15 N-HSQC spectra of 13 of the samples. All metabolites were found to be small molecules that could be separated readily from the labelled proteins by dialysis. No significant transamination activity was observed except for [ 15 N]Asp, where an enzyme in the cell extract efficiently converted Asp fi Asn. This activity was suppressed by replacing the normally high levels of potassium glutamate in the reaction mixture with ammonium or potassium acetate. In addition, the activity of peptide deformylase appeared to be generally reduced in the cell-free expression system.

Translational incorporation of L‐3,4‐dihydroxyphenylalanine into proteins

“…The larger component corresponds to a form of the protein that retains the N-formyl group on the N-terminal methionine residue (calculated M r 19 249.6), and the smaller is the mature protein produced after deformylation (calculated M r 19 221.6). This is in accord with the results of a previous NMR study, in which amide resonances could be observed for the N-terminal methionine as well as for the following residue in hCypA [18], indicating that our S30 extract is deficient in peptide deformylase activity [6].…”

“…1EMA) [22]. The first two proteins had previously been shown to be produced in good yield in the in vitro reaction [5,6,18,23].…”

“…If translational (ribosome-mediated) incorporation of DOPA is a distinct possibility, the structural and functional consequences of DOPA incorporation would be important to assess. This study used a preparative E. coli cell-free transcription ⁄ translation system [5,18] to incorporate DOPA into four different in vitro-synthesized proteins. MS and NMR spectroscopy were used to verify whether DOPA incorporation occurred at positions normally occupied by tyrosine, and to assess the level of DOPA incorporation.…”

Incorporation of chlorinated analogues of aliphatic amino acids during cell-free protein synthesis

“…2 These approaches have been used for the synthesis of proteins containing high levels of fluorinated, 5 dehydro 6 and isotopically-labelled 7,8 amino acids, as well as 3,4-dihydroxy-Phe 9 and seleno-Met 10 in place of Tyr and Met, respectively.…”

“…7,9,20 The native protein was prepared using each of the 20 normal amino acids. Incorporation of other amino acids was investigated through their substitution for one of the 20.…”