Membrane proteins(MPs)have been widely studied in structural,functional proteomics and pharmaceutical research.However,very little is known about the structurefunction relationship due to diffculties in producing MPs by recombinant methods.Chemical protein synthesis(CPS)can offer MPs with diverse predesigned changes with the atom-by-atom precision for advanced biophysical studies.A great challenge for CPS is the difficulty of preparing and handling the hydrophobic transmembrane segments during both solid-phase peptide synthesis(SPPS)and ligations.Here we report a general method for total chemical synthesis of small to medium-sized MPs at multi-milligram scale via Fmoc-SPPS.Its efficiency and practicality is demonstrated by the successful synthesis of Ser64-phosphorylated influenza A virus M2 proton channel and the membrane-bound domain of an inward rectifier K+ channel protein Kir5.1 with a single channel activity.