Computational protocol: Enhanced Yield of Recombinant Proteins with Site-Specifically Incorporated Unnatural Amino Acids Using a Cell-Free Expression System

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[…] The trypsin cleavage sites in WT GFP were predicted by PeptideCutter ( In-gel digestion of purified GFP containing UAA, was performed using Trypsin Gold (Promega, Madison, WI) according to a modified manufacturer’s procedure. Briefly, the gel bands corresponding in size to GFP were finely sliced, washed for 5 minutes with water, 15 minutes with acetonitrile (ACN), and dried in a vacuum centrifuge. After reduction (10 mM dithiothreitol in 100 mM NH4HCO3) and alkylation (10% iodoacetamide in 100 mM NH4HCO3), dried gel slices were incubated on ice for 15–30 minutes with Trypsin gold (12.5 ng/µL) in digestion buffer (50 mM NH4HCO3, 5 mM CaCl2). Excess buffer was removed and 20–30 µL of digestion buffer without Trypsin Gold was added to the gel slices followed by incubation at 37°C overnight. Peptides were extracted from the gel slices by washing once with 25 mM NH4HCO3, ACN and 1% formic acid (FA). The samples were then dried in a vacuum centrifuge. The extracted peptides were purified and concentrated using ZipTip pipette tips (Millipore), following the manufacturer’s instruction.ESI-MS/MS analysis was performed using reverse phase nano-LC (Agilent Technology) connected directly to the LTQ XL Orbitrap EDT mass spectrometer (Thermo Electron, Wien, Austria) at the Analytical Research Services & Instrumentation Unit, BGU. The peptides were eluted with an increasing ACN gradient (Solvent A, 0.1% FA, 5% ACN; Solvent B, 0.1% FA, 80% ACN) over a period of 70 min. MS/MS spectra were acquired in a data-dependent fashion. Instrument control was performed using the Xcalibur software package (Thermo Electron).Theoretical monoisotopic masses for the peptides generated by trypsin digestion of WT GFP were predicted with PeptideMass (, while fragmentation of the FSVSGEGEGDATYGK peptide and theoretical molecular masses of the peptide species were calculated with the MS-Product software at the ProteinProspector web service ( Theoretical molecular masses for peptides containing UAA were adjusted manually. […]

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