Fig. 4.

Multi-site ncAA incorporation at high yield and purity. a Schematic of the protein sequences for wild-type ELPs containing three pentapeptide repeats per monomer unit (ELP-WT) and ELPs containing 1 ncAA per monomer unit (ELP-UAG). b SDS-PAGE and autoradiogram analysis of cell-free produced ELP-WT and ELP-UAG 20-, 30-, and 40-mers in the presence ( + ) and absence ( – ) of p-acetyl-L-phenylalanine (pAcF). Numbers next to the molecular weight ladder (L) represent the approximate kilodalton (kDa) size of the band. c Total protein yields of cell-free synthesized ELPs (20-, 30, and 40-mers) after incubation at 30 °C for 20 h are shown. Three independent batch CFPS reactions were performed for each sample (n = 3). Error bar = 1 SD. d–f Deconvoluted mass spectra of ELPs obtained by top-down mass spectrometry illustrate complete, site-specific incorporation of pAcF at d 20, e 30, and f 40 sites. Deconvoluted average masses for the major peaks in each spectrum (Exper) match the theoretical average mass (Theor) for each species within 1.2 Da. Smaller peaks next to the major peaks arise from minor oxidation of the protein during electrospray ionization