Chemical synthesis of membrane proteins by the removable backbone modification method

Nat Protoc. 2017 Dec;12(12):2554-2569. doi: 10.1038/nprot.2017.129. Epub 2017 Nov 16.


Chemical synthesis can produce membrane proteins bearing specifically designed modifications (e.g., phosphorylation, isotope labeling) that are difficult to obtain through recombinant protein expression approaches. The resulting homogeneously modified synthetic membrane proteins are valuable tools for many advanced biochemical and biophysical studies. This protocol describes the chemical synthesis of membrane proteins by condensation of transmembrane peptide segments through native chemical ligation. To avoid common problems encountered due to the poor solubility of transmembrane peptides in almost any solvent, we describe an effective procedure for the chemical synthesis of membrane proteins through the removable-backbone modification (RBM) strategy. Two key steps of this protocol are: (i) installation of solubilizing Arg4-tagged RBM groups into the transmembrane peptides at any primary amino acid through Fmoc (9-fluorenylmethyloxycarbonyl) solid-phase peptide synthesis and (ii) native ligation of the full-length sequence, followed by removal of the RBM tags by TFA (trifluoroacetic acid) cocktails to afford the native protein. The installation of RBM groups is achieved by using 4-methoxy-5-nitrosalicyladehyde by reduction amination to incorporate an activated O-to-N acyl transfer auxiliary. The Arg4-tag-modified membrane-spanning peptide segments behave like water-soluble peptides to facilitate their purification, ligation and mass characterization.

MeSH terms

  • Amino Acid Sequence
  • Fluorenes / chemical synthesis
  • Fluorenes / chemistry
  • Membrane Proteins / chemical synthesis*
  • Membrane Proteins / chemistry
  • Peptides / chemical synthesis*
  • Peptides / chemistry
  • Solid-Phase Synthesis Techniques / methods*
  • Trifluoroacetic Acid / chemical synthesis
  • Trifluoroacetic Acid / chemistry


  • 9-fluorenylmethoxycarbonyl
  • Fluorenes
  • Membrane Proteins
  • Peptides
  • Trifluoroacetic Acid